Monoclonal antibodies recognizing oval cells induced in the liver of rats by N-2-fluorenylacetamide or ethionine in a choline-deficient diet

In this paper, we describe the production of a panel of monoclonal antibodies which define antigens which distinguish between hepatocytes and oval cells. These antibodies were obtained from hybridomas constructed from the spleens of mice immunized by a novel protocol designed to suppress response to unwanted or immunodominant epitopes. Of the antibodies obtained, four, 258.7, 270.11, 258.34, and 270.38, were directed to antigens of morphologically defined oval cells, while two, 258.26 and 270.26, defined cytoplasmic antigens of hepatocytes. Examination of frozen sections of normal, regenerating adult and fetal liver and livers from rats fed 2-acetylaminofluorene or ethionine in a choline-deficient diet indicates that morphologically defined oval cells may in fact comprise a phenotypically complex set of cells composed of at least three antigenically distinct subpopulations. The patterns of expression of the antigens defined by these antibodies suggest two possible pathways of liver cell differentiation.     

Azaserine carcinogenesis: organ susceptibility change in rats fed a diet devoid of choline.

In rats, azaserine is primarily a pancreatic carcinogen and only induces hepatomas with a very low incidence. We have investigated the effects of feeding a choline-devoid (CD) diet upon the carcinogenicity of azaserine. Groups of male Wistar rats were fed a CD or a choline-supplemented (CS) diet. Azaserine (30 mg/kg in 2 ml of saline) was injected IP twice a week for the first 4 weeks, and once a week thereafter, for a total of 14 injections. Control groups were fed the CD or CS diet and were injected similarly with saline. Four to seven animals from each group were killed, 1, 4 and 6 months after the first azaserine injection, and the liver and pancreas were examined histologically. None of the control animals fed the CD or CS diet and given saline injections developed tumors of the liver or pancreas. No hepatomas were observed in 12 rats fed the CS diet and treated with azaserine. On the other hand, 3 of 5 and 5 of 7 rats killed after 4 and 6 months, respectively, of treatment with azaserine and the CD diet showed multiple hepatomas. Rats treated with azaserine developed multiple atypical acinar cell nodules (AACN) of the pancreas after 4 months irrespective of whether choline was present or absent in the diet. However, the number of AACN in rats fed the CD diet was significantly smaller than in rats fed the CS diet. It is concluded that a diet devoid of choline changes the organ susceptibility to the carcinogenic action of azaserine, enhancing hepatocarcinogenesis and reducing the action of the carcinogen on the pancreas.     

Distribution of alpha-fetoprotein- and albumin-containing cells in the livers of Fischer rats fed four cycles of N-2-fluorenylacetamide.

Immunofluorescent localization of alpha-fetoprotein (AFP)- and albumin-containing cells was determined in the livers of Fischer rats fed 0.05% N-2-acetylfluorenamide for four 2-weeks-on, 1-week-off cycles. After this exposure multiple changes in the liver include over 1000 neoplastic nodules/liver, as well as extensive production of so-called oval cells and focal zones of atypical hepatocellular hyperplasia. Approximately 1% of the oval cells contain AFP, and about half of the zones of atypical hyperplasia include cells that contain AFP, but none of the neoplastic nodules or normal hepatocytes have any AFP-containing cells. Since up to 60% of the hepatocellular carcinomas developing from this regimen will predictably produce AFP, it is tentatively concluded that hepatocellular carcinoma may arise not only from "premalignant" neoplastic nodules but also from oval cells or the atpyical differentiation of hepatocytes.     

Expression of L- and M2-pyruvate kinases in proliferating oval cells and cholangiocellular lesions developing in the livers of rats fed a methyl-deficient diet

Male outbred Sprague-Dawley rats were fed a cholinedeficientdiet containing 0.1% w/w DL-ethionine (CDE) for up to 22 weeks.The expression of the pyruvate kinase isoenzymes L (L-PK) andM₂ (M₂ was immunohistochemically analyzed in liver slices fromrats killed 4, 10, 14 and 22 weeks after starting the treatment.M₂ was detected in bile duct epithelial cells of untreated ratsand in proliferating oval cells, cholangiofibroses and cholangiofibromasof CDE-fed animals. Thus, M₂ can be viewed as a positive markerof the bile duct epithellial/oval cell compartment. L-PK, aparenchymal cell-specific protein in untreated rat liver, wasnot present In proliferating oval cells, but was consistentlyobserved In cells that were part of the ductal structures inthe cholangiofibroses and cholanglofibromas. Based on theirmorphology, the L-PK-posltlve duct cells were undoubtedly partof the bile duct epithellal cell lineage and no L-PK-positivehepatocyte-like cells were observed in the ducts. Hence, thisstudy clearly shows that the mere presence of a liver parenchymalcell marker in cells of the bile duct epithelial/oval cell compartmentdoes not necessarily preclude that these cells are undergoinga differentiation into preneoplastic parenchymal cells, as haspreviously been suggested.     

Enzyme histochemical and immunohistochemical characterization of oval and parenchymal cells proliferating in livers of 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 for up to 30 weeks. Liver slices from rats killed 4, 6, 10, 14, 22 and 30 weeks after starting the treatment were histochemically analyzed for the following parameters: basophilia, expression of cytokeratin 19 (which in the liver is bile duct epithelial cell-specific), glycogen content and activities of glycogen synthetase (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 diet induced necrosis of single parenchymal cells and a massive proliferation of oval cells within 4-6 weeks; thereafter cholangiofibroses, cystic cholangiomas and some cholangiofibromas, but no cholangiocarcinomas, were observed. Oval cells, cholangiofibroses, cystic cholangiomas and cholangiofibromas expressed cytokeratin 19, whereas parenchymal cells, foci of altered hepatocytes and hepatocellular adenomas did not; this observation does not support a precursor-product relationship between oval and parenchymal cells. SYN, PHO, G6PASE, G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT activities were detected in oval cells; cholangiofibrotic lesions, cystic cholangiomas and cholangiofibromas stained strongly for GAPDH, G3PDH and MDH. In livers from rats fed the diet for 10 weeks, single hepatocytes storing high amounts of glycogen appeared in the parenchyma. There was no indication of a transition from the oval cell population to hepatocytes storing glycogen in excess. Foci of glycogen-storing cells were scattered all over the lobes after 14 and 22 weeks; they had increased G6PASE, G6PDH, ALKPASE and GGT activities. Mixed cell foci and hepatocellular adenomas developed within 22-30 weeks and exhibited a remarkable decrease of G6PASE activity, a strong increase of G6PDH, GAPDH, G3PDH and MDH activities as well as extremely high ALKPASE and GGT activities. The data support the concept that during hepatocarcinogenesis, a number of sequential changes in the activities of various enzymes involved in carbohydrate metabolism occur and that a correlation between morphology and enzyme pattern in the focal lesions does in fact exist. Furthermore, our results suggest that two different cell lineages are involved in the development of cholangiocellular tumors from oval cells and hepatocellular tumors from hepatocytes.     

-Fetoprotein and hepatocarcinogenesis in rats fed 3'-methyl-4-(dimethylamino)azobenzene or N-2-fluorenylacetamide

Serum α-fetoprotein (af) of rats during hepatocarcinogenesis by 3′-methyl-4-(dimethylamino)azobenzene (3′-Me-DAB) or N-2-Fluorenylacetamide (FAA) was checked by the micro-Ouchterlony method throughout the course of the study. The serum af appeared in two phases, early from the 3rd to 5th week and late during the development of large carcinomas. The frequency of early appearance of serum af varied considerably according to the difference in age, sex, and strain of the animals or the difference in type or dose of the carcinogen. In general, 3′-Me-DAB was a much stronger carcinogen than FAA in producing early serum af. A good correlation was observed between the serum af level and the intensity of oval cell proliferation in the liver at this stage. The cell composing the nodular hyperplasia in 3′-Me-DAB or the area of hyperplasia in FAA carcinogenesis, which replaced the major part of the liver in the next stage and had been considered the most possible forerunner of the carcinoma, did not seem to produce af. The serum af in the late phase was produced by the carcinoma. The majority of the 3′-Me-DAB-induced carcinomas belonged to the mixed type carcinoma. This type of carcinoma was strongly af-producing with the required minimal size of 10 mm in diameter for positive serum af. Most of the FAA-induced carcinomas were of the trabecular type. This type of carcinoma was relatively weak in af-production and the serum af was negative until a trabecular carcinoma became larger than 20 mm in diameter. The appearance of early serum af was distinct evidence of a fair progress of hepatocarcinogenesis, and a positive correlation was found between the degree of this phenomenon and the frequency of development of a carcinoma producing large amounts of af. However, the appearance of early serum af was not an absolute requisite for later development of carcinomas.     

Comparison of oval cells induced in rat liver by feeding N-2-fluorenylacetamide in a choline-devoid diet and bile duct cells induced by feeding 4,4'-diaminodiphenylmethane

Oval cells and duct-like structures produced in the livers of rats fed N-2-fluorenylacetamide in a choline-devoid diet differ from bile ducts produced after feeding 4,4'-diaminodiphenylmethane. Rapid elevations of serum alpha-fetoprotein (AFP) occur after feeding N-2-fluorenylacetamide in a choline-devoid diet; no elevations of AFP are seen during 4,4'-diaminodiphenylmethane feeding. The duct-like structures associated with oval cells frequently contain AFP and albumin and are faintly delineated by laminin, whereas normal bile ducts and ducts induced by 4,4'-diaminodiphenylmethane do not contain AFP or albumin and are delineated by an intensely staining layer of laminin. Zones of oval cell proliferation label intensely for fibronectin, whereas zones of bile duct proliferation label much less intensely. It is concluded that the "tubuloform degeneration" seen after carcinogen exposure does not necessarily represent differentiation to true bile duct structures and that oval cells may neither derive from nor differentiate into bile ducts. Oval cells have characteristics more like fetal hepatocytes than ductular cells and may represent a "stem cell"-like population with potential for loss of growth control and malignant transformation.     

Microsomal metabolism of the carcinogen, N-2-fluorenylacetamide, by the mammary gland and liver of female rats. I. Ring- and N-hydroxylations of N-2-fluorenylacetamide

We determined ring- and N-hydroxybtions of a systemic mammarygland cardnogen, N-2-fluorenylaeetamide (2-FAA), by microsomalfractions of liver and mammary gland of female rats and theeffects of in vivo and/or in vitro modifiers of these oxidations.Pretreatment of lactating rats with 3-methylcholanthrene (3-MC)or ß-naphthoflavone (ß-NF) and non-lactating(50-day old virgin) rats with ß-NF showed similareffects in that the formation of 3-, 5-, 7-, 9- and N-hydroxy-2-FAAby hepatic microsomes was increased manyfold and the formationof 1-hydroxy-2-FAA was induced. In mammary gland microsomes,the formation of 3-, 5- and 7-hydroxy-2-FAA was likewise increased,but of 9-hydroxy-2-FAA was unaffected. Only mammary microsomesof lactating rats had capacity for N-hydroxylation which wasincreased {small tilde}3 times by pretreatment of rats with3-MC or ß-NF. All of the induced increases of metabolitesof 2-FAA in hepatic and mammary microsomes were inhibited by0.1 mM -naphthoflavone (-NF) in vitro. Pretreatment of non-lactatingrats with phenobarbital increased only the formation of 7-hydroxy-2-FAAin hepatic microsomes which was further stimulated by -NF invitro. The latter also stimulated the formation of 7- and 9-hydroxy-2-FAA by hepatic microsomes of the uninduced rats, buthad no effects in mammary microsomes, in which 9-hydroxy-2-FAAwas a major metabolite. Hence, the data showed qualitative andquantitative differences between lactating and non-lactatingrats in metabolism of 2-FAA by mammary microsomes which mayresult from differences in the levels (e.g., of cytochrome P-450)and activities of microsomal enzymes determined herein. In hepaticmicrosomes of these rats, differences in quantities of metabolitesof 2-FAA (3-, 7-, 9- and N-hydroxy-2-FAA) were found in cornoil-treated rats only. The solvent (methanol or acetone) usedfor addition of 2-FAA to the incubation mixtures altered quantitativelythe metabolite profiles in hepatic and mammary microsomes of3-MC or ß-NF treated rats. The formations of 1- and3- or 5- and 7-hydroxy-2-FAA were greater in the presence ofacetone or methanol, respectively. The results of this studysuggest that the formation of phenolic and N-hydroxy metabolitesof 2-FAA in both hepatic and mammary microsomes of lactatingrats is catalyzed by similar form(s) of cytochrome P-450 inducedby pretreatment with 3-MC or ß-NF. Lack of inductionN-hydroxylation of 2-FAA in mammary microsomes of non-lactatingrats supports our earlier conclusion that the formation of aproximate metabolite in mammary tumorigenesis by 2-FAA is accomplishedin the liver.     

Alterations in expression and methylation of specific genes in livers of rats fed a cancer promoting methyl-deficient diet

We have reported earlier that hypomethylated DNA is rapidly induced in the livers of male Fischer rats fed an extremely methyl-deficient diet (MDD). The early effects of dietary methyl deficiency on the expression of several genes in the livers of such animals have now been investigated. Poly(A)+ RNA was isolated from the livers of rats fed MDD or a similar diet supplemented with adequate supplies of choline, methionine, folic acid and vitamin B12 (CSD) for periods ranging from 1 to 4 weeks. The levels of mRNAs for the c-myc and c-fos protooncogenes in livers of rats given either MDD or the liver carcinogen, 2-acetylaminofluorene (AAF), were compared by Northern blot analysis with those in livers of animals given control diets. Both AAF and MDD induced significant elevations in levels of mRNAs specific for these two genes. After 1 week of MDD intake, large increases in the levels of c-myc and c-fos mRNAs and a smaller increase in the levels of c-Ha-ras mRNAs were observed. In contrast, there were marked decreases in the levels of mRNAs for epidermal growth factor receptor and for epidermal growth factor. These effects on mRNA accumulation persisted and were further enhanced during a 4 week period of MDD feeding. The appearance of hypomethylated DNA in the livers of these MDD-fed rats coincided with the observed changes in levels of mRNA for these genes associated with the regulation of cell growth. Increases in levels of mRNA for c-fos, c-Ha-ras and c-myc were correlated with loss of methylation at specific sites within these genes as early as 1 week after the start of MDD feeding. These combined observations are consistent with the hypothesis that methyl-deficient diets are cancer promoting and/or carcinogenic, at least in part, because they induce hypomethylation of DNA with concomitant alterations in the regulation of gene expression.     

Elevated levels of prostaglandin E2 in the liver of rats fed a choline deficient diet: possible involvement in liver tumor promotion

The effect of feeding a choline deficient (CD) diet, an efficient liver tumor promoting regimen, on the prostaglandin metabolism in the liver of male Sprague-Dawley rats was investigated. The possible biological significance of the alteration was examined using hypolipidemic peroxisome proliferators and modifiers of prostaglandin metabolism such as indomethacin and menhaden oil in the short term assay of the induction of enzyme altered foci in the liver. A CD diet, when fed for 10-30 days, induced 2-2.5 times increases in the levels of prostaglandin E2 (PGE2) in the liver, while the hypolipidemic peroxisome proliferators, 4-chloro-6(2,3-xylidino) pyrimidinylthio(N-hydroxyethyl)-acetamide (BR931) and di(2-ethylhexyl)-phthalate (DEHP), markedly reduced the levels of this metabolite. The addition of BR931 or indomethacin to a CD diet suppressed the diet-induced elevations of PGE2 and a substitution of fats in a CD diet with menhaden oil had the same effect. Furthermore, both indomethacin and menhaden oil added to a CD diet suppressed the induction of gamma-glutamyltranspeptidase positive hepatocyte foci in the liver of rats initiated with a single dose of diethylnitrosamine after 8 weeks of the dietary promotion. The results suggest that altered prostaglandin metabolism may be involved in the liver tumor promoting effect of a CD diet.     

Microsomal metabolism of the carcinogen, N-2-fluorenylacetamide, by the mammary gland and liver of female rats. II. Glucuronidation of ring- and N-hydroxylated metabolites of N-2-fluorenylacetamide

We determined UDP-glucuronyltransferse (UDP-GT) activities ofhepatic and mammary gland microsomes of female rats with p-nitrophenoland the ring- and N-hydroxylated metabolites of N-2-fluorenylacetamlde(2-FAA) and the effects of hepatic inducers of UDP-GT's on theseglucuronidations. Pre-treatment of non-lactating (NL)and lactating(L) rats with ß-naphthoflavone (ß-NF) significantlyincreased glucuronidations, of p-nitrophenil, aphenolic metabolitesof 2-FAA, especially of 5-hydroxy 2-FAA and also of N-hydrosy-2-FAAby hepatic microsomes. Pre-treatment of L rats with ß-NFor 3-methyl cholanthrene (3-MC) significantly incresed glucuronidationsof these compounds by mammary gland microsomes suggesting thatboth liver and mammary gland of L rats possess similar UDP-GTactivities. In NL rats, UDP-GT activities of mammary microsomestoward phenols were greater than in L rats, and except for thatof 5- and 7- hydroxy-2-FAA, were not inducible with ß-NF.The data obtained with L rats, the greater magnitude of stimulationof the hepatic UDP-GT of NL rats by ß-NF than by phenobarbital, and the lack of effect of the latter on UDP-GT ofmammary microsomes suggested that the phenolic metab olitesof 2-FAA and N-hydroxy-2-FAA share chiefly the characteristicsof substrates for group 1 UDP-GT activities (i.e., those induciblewith ß-NF or 3-MC). Neither inducer increased glucuronidationof 9-hydroxy-2-FAA, a relatively poor substrate for UDP-GT ofmammary or hepatic micro somes. In contrast to hepatic microsomeswhich formed considerable amounts of the glucuronide of N-hydroxy-2-FAA,mammary gland microsomes glucuronidated this substrate to aminor extent only. This suggested that glu curonide of N-hydroxy-2-FAAmay play a role in systemic, but not in local mammary tumorigenesisby N-hydroxy-2-FAA.