COMPARATIVE STUDY OF ABNORMALITY IN GLYCOGEN STORING CAPACITY AND OTHER HISTOCHEMICAL PHENOTYPIC CHANGES IN CARCINOGEN-INDUCED HEPATOCELLULAR PRENEOPLASTIC LESIONS IN RATS

A sequential comparison was made between abnormal glycogen storage and other histochemical phenotypic changes in hepatocellular precancerous lesions (altered foci and neoplastic nodules) during various stages in the process of development of cancer in rat liver. N-2-fluorenylacetamide was fed to male rats for 8 weeks and groups of rats were killed at the end of carcinogen feeding and at 12 and 24 weeks on control diet. Foci rich in glycogen storage accounted for a majority of all foci over the course of experiment, while foci devoid of glycogen storage, which were absent at the end of carcinogen feeding, gradually increased in number during maintenance. Glycogen-deficient lesions that might appear to arise from glycogen-rich lesions displayed hyper-basophilia demonstrated by toluidine blue reaction, but often lacked gamma-glutamyl transpeptidase activity. Resistance to iron accumulation was consistently shown in all precursor lesions for hepatocellular carcinoma in the siderotic liver regardless of abundance or absence of cellular glycogen. It was suggested that properties such as loss of glycogen storing capacity, hyperbaso-philia, and some cellular atypicality resembling those of carcinoma cells might be essential elements for malignant progression.     

Comparative study of abnormality in glycogen storing capacity and other histochemical phenotypic changes in carcinogen-induced hepatocellular preneoplastic lesions in rats

A sequential comparison was made between abnormal glycogen storage and other histochemical phenotypic changes in hepatocellular precancerous lesions (altered foci and neoplastic nodules) during various stages in the process of development of cancer in rat liver. N-2-fluorenylacetamide was fed to male rats for 8 weeks and groups of rats were killed at the end of carcinogen feeding and at 12 and 24 weeks on control diet. Foci rich in glycogen storage accounted for a majority of all foci over the course of experiment, while foci devoid of glycogen storage, which were absent at the end of carcinogen feeding, gradually increased in number during maintenance. Glycogen-deficient lesions that might appear to arise from glycogen-rich lesions displayed hyperbasophilia demonstrated by toluidine blue reaction, but often lacked gamma-glutamyl transpeptidase activity. Resistance to iron accumulation was consistently shown in all precursor lesions for hepatocellular carcinoma in the siderotic liver regardless of abundance or absence of cellular glycogen. It was suggested that properties such as loss of glycogen storing capacity, hyperbasophilia, and some cellular atypicality resembling those of carcinoma cells might be essential elements for malignant progression.     

Abnormalities in liver iron accumulation during N-2-fluorenylacetamide hepatocarcinogenesis that are dependent or independent of continued carcinogen action

The characteristics of liver iron accumulation were studied during N-2-fluorenylacetamide (FAA)-induced hepatocarcinogenesis in rats. After injection of iron-dextran in control rats, hepatocytes accumulated stainable iron evenly throughout hepatic lobules. During the feeding of FAA, iron accumulation was reduced in the midzonal and centrilobular regions. After FAA removal, hepatocytes in these regions again accumulated high amounts of iron. Hepatocellular altered foci induced by FAA displayed rather uniform (> 94%) iron-exclusion during FAA feeding. After FAA removal, however, iron-exclusion was lost in a fraction of the foci, while others (40-64%) remained resistant to iron accumulation. A large majority of liver neoplasms (> 93%) displayed resistance to cellular iron accumulation both during FAA feeding and after removal of FAA. Thus, iron-exclusion by liver neoplasms is carcinogen-independent and irreversible, in contrast with that of normal hepatocytes which is completely carcinogen-dependent and reversible. Altered foci appear to represent two populations: one is characterized by reversible iron-exclusion whereas the other, like neoplasms, possesses permanent iron-exclusion.     

Histogenesis of dieldrin and DDT-induced hepatocellular carcinoma in Balb/c mice

Male, Balb/c mice were fed diets containing dieldrin (10 ppm) and DDT (100-175 ppm) for 75 weeks. Control and treated mice were serially killed and their livers analyzed by histological and histochemical procedures after 2, 4, 8, 16, 36, 52 and 75 weeks of exposure. Mice administered both chlorinated hydrocarbons initially responded with centrolobular hepatocytomegaly. The cells were characterized by decreased glucose-6-phosphatase and succinate dehydrogenase activity. At later periods 52 through 75 weeks, foci of phenotypically-altered hepatocytes were noted. The cells of these lesions were basophilic or clear-staining in hematoxylin and eosin-stained sections and displayed increased gamma glutamyl transpeptidase activity. In mice preloaded with iron dextran, cells of foci were negative for iron when the surrounding parenchyma was siderotic. Hepatocellular adenomas (HA) and carcinomas (HPC) were composed of cells with increased gamma glutamyl transpeptidase and glucose-6-phosphate dehydrogenase and decreased glucose-6-phosphatase and succinate dehydrogenase activity. In iron loaded mice, the cells of HA and HPC did not stain for iron in otherwise siderotic surroundings. Both hepatocellular foci and adenomas may be potential precursors of mouse hepatocellular carcinomas.     

γ-Glutamyl transpeptidase-dependent mutagenicity and cytotoxicity of γ-glutamyl derivatives: A model for biochemical targeting of chemotherapeutic agents

Many carcinomas in humans are rich in γ-glutamyl transpeptidase (GGT), a plasma membrane enzyme that reacts with extracellular substrates. Thus, biochemical targeting of chemotherapeutic agents may be achieved by converting anticancer drugs into their γ-glutamyl derivatives. Chemical conversion of phenylhydrazine (PH) and biochemical modification of daunomycin (DM) into their γ-glutamyl derivatives γ-glutamyl phenylhydrazine (GGPH) and γ-glutamyl DM (GGDM) resulted in the abolishment of their mutagenicity and cytotoxicity, as judged by decreased viability and increased mutant yields in cultures of several Salmonella Ames strains. Commercial γ-glutamyl-p-nitroanilide (GGPNA) was not toxic or mutagenic. Mutagenicity and/or cytotoxicity of these γ-glutamyl derivatives were restored upon reaction with GGT, with concomitant release of PH, and p-nitroaniline (PNA). The GGT-dependent release of DM from GGDM was demonstrated by thin layer chromatography (TLC), spectral analysis, and specific mutagenicity. Mutagenicity and/or cytotoxicity of γ-glutamyl derivatives increased in the presence of glycylglycine, a GGT activator, and decreased in the presence of serine-borate, a GGT inhibitor. GGDM retained considerable DNA binding capacity. Its inability to kill and mutagenize was due to altered transport properties. The results are compatible with the notion that γ-glutamylation is a feasible method for biochemical targeting of drugs containing a primary amino group to GGT-rich tumors. Environ. Mol. Mutagen. 32:377–386, 1998 © 1998 Wiley-Liss, Inc.     

Mesenchymal-epithelial transformation of Ito cells in vitro

Cultured pure population of Ito cells isolated from adult rat liver expressed epithelial markers cytokeratin-8, α-fetoprotein, and γ-glutamyl transpeptidase after forming a dense monolayer. Mesenchymal-epithelial transformation of these cells is possible, which suggests them as candidates of hepatic stem cells. __________ Translated from Kletochnye Tekhnologii v Biologii i Medicine, No. 3, pp. 150–153, August, 2006     

The sensitivity and heterogeneity of histochemical markers for altered foci involved in liver carcinogenesis.

Subcutaneous injection of iron dextran resulted in a hepatic siderosis within 2 weeks in rats, as previously reported for mice. Hepatic carcinomas as well as neoplastic nodules in rats were entirely or mainly free of stainable iron and, thus, could be readily identified histologically. In addition, early carcinogen-induced altered foci were resistant to iron accumulation. In rats fed 0.02% N-2-fluorenylacetamide (FAA) for 13 weeks, the number of iron-resistant foci identified following iron injection was the same as that observed with dietary iron overload. Histochemical investigation of enzymatic markers that have been used to identify foci in rats revealed that foci characterized by enzymatic reactions of positive gamma-glutamyl transpeptidase and decreased adenosine triphosphatase and glucose-6-phosphatase corresponded to those characterized by resistance to iron accumulation. However, in quantitative analysis of the early carcinogen-induced foci in rats given iron dextran following a diet containing 0.02% 2-FAA for 13 weeks, more lesions were detected by resistance to iron accumulation than by any of these other properties. There was considerable phenotypic heterogeneity among foci for the enzyme markers. It is concluded that resistance to iron accumulation is a more sensitive and reliable marker for early carcinogen-induced altered hepatocellular foci than is any other histochemical property.     

Sequential analysis of hepatic carcinogenesis: a comparative study of the ultrastructure of preneoplastic, malignant, prenatal, postnatal, and regenerating liver.

The objective of this study was to compare the fine structure of presumptive preneoplastic hepatocytes at various times during liver carcinogenesis with that of normal, developing, and regenerating liver and of hepatocellular carcinomas, using transmission and scanning electron microscopy. A new model of liver carcinogenesis was used in which several of the early steps are quite well synchronized. A single initiating dose of diethylnitrosamine induced isolated islands of altered hepatocytes. The cells were characterized by persistence of glycogen despite starvation, increase in smooth endoplasmic reticulum, and hypertrophic nucleoli. Following intense selection of the altered hepatocytes by dietary 2-acetylaminofluorene plus partial hepatectomy, the affected hepatocytes proliferated rapidly to produce basophilic foci. These early hyperplastic lesions revealed stellate-shaped dilated bile canaliculi lined by blebs and abnormally thick elongated microvilli, a decreased number of microvilli on the sinusoidal surface, a marked increase in smooth endoplasmic reticulum, large nucleoli, and bundles of pericanalicular microfilaments. A majority of the proliferating lesions reacquired a normal organizational pattern within several weeks after partial hepatectomy and could not be distinguished from normal liver. A small number continued to grow and become typical persistent hyperplastic nodules. These showed significant widening of intercellular spaces between hepatocytes, elongated microvilli over large regions of the cell surface, many invaginations of the cell membrane, and irregularly shaped bile canaliculi. Sequential changes in focal hyperplastic hepatocytes during carcinogenesis could be distinguished from normal, developing, and regenerating liver. The major differences involved the cell surfaces and cytoplasmic organelles. The findings are compatible with the hypothesis that a carcinogen may act by inducing alterations in a small number of hepatocytes and that hepatocellular carcinomas arise through stepwise evolutional changes in these cells.     

Tomato oleoresin inhibits DNA damage but not diethylnitrosamine-induced rat hepatocarcinogenesis

Various studies have shown that lycopene, a non-provitamin A carotenoid, exerts antioxidant, antimutagenic and anticarcinogenic activities in different in vitro and in vivo systems. However, the results concerning its chemopreventive potential on rat hepatocarcinogenesis are ambiguous. The aim of the present study was to investigate the antigenotoxic and anticarcinogenic effects of dietary tomato oleoresin adjusted to lycopene concentration at 30, 100 or 300 ppm (administered 2 weeks before and during or 8 weeks after carcinogen exposure) on liver of male Wistar rats treated with a single intraperitoneal dose of 20 or 100mg/kg of diethylnitrosamine (DEN), respectively. The level of DNA damage in liver cells and the development of putative preneoplastic single hepatocytes, minifoci and foci of altered hepatocytes (FHA) positive for glutathione S-transferase (GST-P) were used as endpoints. Significant reduction of DNA damage was detected when the highest lycopene concentration was administered before and during the DEN exposure (20mg/kg). However, the results also showed that lycopene consumption did not reduce cell proliferation in normal hepatocytes or the growth of initiated hepatocytes into minifoci positive for GST-P during early regenerative response after 70% partial hepatectomy, or the number and area of GST-P positive FHA induced by DEN (100mg/kg) at the end of week 10. Taken together, the data suggest a chemopreventive effect of tomato oleoresin against DNA damage induced by DEN but no clear effectiveness in initiating or promoting phases of rat hepatocarcinogenesis.     

Iron and the liver: subcellular distribution of iron and decreased microsomal cytochrome P-450 in livers of iron-loaded rats.

To understand better the intracellular iron distribution and metabolic consequences of chronic hepatic iron overload, rats were given large doses of iron dextran or ferric citrate intraperitoneally. They accumulated large quantities of iron within Kupffer cells and hepatocytes. The relative subcellular iron distributions were similar in controls and iron-loaded rats, despite a ten- to 20-fold difference in hepatic iron concentration. Electron microscopy of whole liver and subcellular particulate fractions suggested that iron was present in highest concentration in lysosomes, which were rendered more labile by its presence. Nevertheless, quantitative iron determinations on all subcellular fractions, obtained by two preparative methods, showed that most of the iron was present in the "soluble" fraction. The amount of iron in the "microsomal" fraction varied, depending on the techniques used for preparation of this fraction. Cytochrome P-450 and total heme concentrations were decreased 40% to 50% in microsomes isolated from iron-loaded livers.     

Placental glutathione S-transferase (GST-P) as a new marker for hepatocarcinogenesis: in vivo short-term screening for hepatocarcinogens

Our laboratory has developed an in vivo short-term screening test for hepatocarcinogens based on quantitation of gamma-glutamyl transpeptidase (gamma-GT) positive foci. However, gamma-GT positive hepatocytes appear in periportal areas under a variety of circumstances apparently unrelated to hepatocarcinogenesis. Glutathione S-transferase placental type (GST-P), which is hardly detectable in normal rat liver, was recently demonstrated as a new marker protein for preneoplastic liver foci. In experiment I, rats were initially given a single dose (200 mg/kg) of diethylnitrosamine intraperitoneally and 2 weeks later were treated with test compounds for 6 weeks. All rats were subjected to partial hepatectomy at week 3. The long-term development of preneoplastic lesions was followed in rats for 50 weeks. The immunohistochemical investigation of GST-P binding and the histochemical demonstration of gamma-GT in serial sections revealed that almost all gamma-GT foci were GST-P positive, but 5-10% of GST-P foci could not be detected by gamma-GT staining. From week 8, many gamma-GT foci partially lost gamma-GT activity. However, no comparable disappearance of GST-P was evident in the lesions. All hepatocellular carcinomas (HC) found at week 50 consisted of GST-P positive HC cells. In contrast, 37.9% (11/29) of HC were negative for gamma-GT. In experiment II (in vivo short-term screening test for hepatocarcinogens), rats were treated in the same manner as for experiment I and killed at week 8. Fifty-eight chemicals were investigated for their potential to modify GST-P positive foci development.(ABSTRACT TRUNCATED AT 250 WORDS)     

FAT-STORING CELLS (ITO'S CELL) OF HUMAN LIVER

An enzyme histochemical study was carried out on fat-storing cells, which are distributed in the space of Disse of mammalian liver. The livers used for study consisted of human autopsy materials demonstrating either acute or chronic circulatory disturbance. Fat-storing cells contain abundant glycogen, and demonstrate a marked γ-glutamyl transpeptidase activity and a weak lactate dehydrogenase activity. It is highly probable that an active glyconeo-genesis rather than glycogenolysis is being carried out in these cells. In acute circulatory disturbance, an elevated alkaline phosphatase activity is found in the wall of sinusoids. This phenomenon is a manifestation of activated capillary endothelial cells participating in the regeneration of liver parenchyma. On the other hand, in chronic congestive liver, elevated γ-glutamyl transpeptidase activity in fat-storing cells is concerned in the proliferation of fibers in the wall of sinusoids, and elevated pressure in sinusoids is considered to be the main factor in its increased activation.     

Liver pathology in patients with primary hemochromatosis--homozygous carriers of C282Y mutation

Liver pathology was studied in 3 patients with primary chemochromatosis. In two cases so-called iron free foci with signs of hepatocytes with feature of dysplasia were found. Many siderosomes were found ultrastructurally in the cytoplasma of hepatocytes. Histological markers of virus infection were absent in a patient with positive serum HbsAg and HCV-Ab. Alcohol did not produce typical histological changes. In this case grave liver reticuloendothelial hemosiderosis typical for secondary hemochromatosis and overloading with iron of spleen pulp according to MR imaging were observed.     

Preneoplastic liver cell foci expansion induced by thioacetamide toxicity in drug-primed mice

Mice primed by feeding griseofulvin or diethyl 1,4-dihydro 1,4,6-trimethyl 3,5-pyridine decarboxylate for 5 months followed by drug withdrawal for 1 month (drug-primed mice) were given thioacetamide intraperitoneally, and the livers were subsequently studied at intervals up to 7 days. The hepatocellular proliferative response was measured by immunostaining for proliferative cell nuclear antigen. Necrosis was followed by measuring ALT. Mallory bodies were identified by immunoperoxidase stains for ubiquitin and cytokeratin. Preneoplastic foci were localized using immunofluorescence stain for glutathione S-transferase (GST mu) and histochemical stain for gamma glutamyl transpeptidase (GGT). The results showed that the preneoplastic foci selectively proliferated and expanded and formed nodules as indicated by quantitation of nuclei stained positive for proliferating cell nuclear antigen after thioacetamide treatment. Data support the hypothesis that the preneoplastic foci consisted of clones of hepatocytes which preferentially express GST mu, GGT and Mallory bodies. These preneoplastic cells selectively proliferate in response to the promoter effects of necrosis-induced liver cell regeneration ("chemical partial hepatectomy").     

Is there more than one critical lesion relevant for experimental liver carcinogenesis?

The present study was designed to determine whether there is one or more than one critical lesion, induced by a carcinogen, relevant for initiation. The experimental approach consisted of administering a non-necrogenic dose of the carcinogen 1,2-dimethylhydrazine 2HCl (100 mg/kg, i.p.) to male Fischer 344 rats (120-140 g) and completing the initiation process by two different methods: (i) induction of liver cell proliferation by partial hepatectomy, or (ii) creation of hypomethylation in DNA by giving 5-azacytidine, an agent that inhibits DNA methylation. The initiated hepatocytes were assayed as gamma-glutamyltransferase positive foci. The rationale for the approach was based on the premise that the two methods used for completing the initiation step might give either the same or a different pattern in the incidence of initiated hepatocytes depending on whether one or more than one lesion was important for initiation, particularly if some of the lesions were allowed to repair before applying the cell proliferative stimulus or administering the 5-azacytidine. The results obtained indicated that 5-azacytidine facilitated the induction of the same number of foci whether given 12 or 96 hours after the carcinogen indicating that the critical lesion involved in this mode of initiation persisted up to at least 96 hours. In contrast, our earlier results showed that there was a reduction in the number of gamma-glutamyltransferase positive foci when partial hepatectomy was delayed beyond 24 hours after the carcinogen administration, indicating that the critical lesion involved in this mode of initiation has a half-life of not more than 24 hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of interaction of 2-acetylaminofluorene with normal liver and carcinogen-induced hepatocyte nodules in vivo and in vitro

This study examines the initial uptake and subcellular distribution of the carcinogen [14C]-2-acetylaminofluorene in liver nodules and normal liver. The route of administration of the carcinogen was intravenously through a peripheral branch of the superior mesenteric vein, intragastrically or intraperitoneally. Tissue distribution was initially dependent on blood flow, but the retention after 5 minutes varied between different tissues according to tissue affinity, high in liver, fat and muscle, low in kidney and brain. The major fraction was retained in the liver. In vitro experiments demonstrated that total levels of [14C]-2-acetylaminofluorene were 8-fold lower in hepatocytes from liver nodules compared with normal liver. The 2-acetylaminofluorene was bound more avidly to 12 to 15 kilodalton cytosolic proteins than to 40 to 50 kilodalton proteins in normal liver and this binding was much less in hepatocyte nodules. The subcellular distribution indicated that the microsomal fractions had a greater specificity than mitochondria, homogenate, or cytosol. This specificity was not due to the lipid content of the fractions. Microsomal fractions from liver nodules had 2-fold less [14C]-2-acetylaminofluorene bound than from normal liver. The carcinogen was bound in cytosolic proteins with a peak 90 minutes after intravenous injection, as compared with a peak for microsomes at 10 minutes. These results lend further support for the concept that the biochemical properties in liver nodules minimize the metabolism of xenobiotics in vivo.     

Mechanistic study of transfection of chitosan/DNA complexes coated by anionic poly(γ-glutamic acid)

Chitosan (CS) has been investigated as a non-viral carrier for gene delivery, but resulting in a relatively low transfection. To address this concern, we developed a ternary system comprised the core of CS/DNA complex and the outer coating of an anionic polymer, poly(γ-glutamic acid) (γ-PGA). In molecular dynamic (MD) simulations, we found that γ-PGA was entangle tightly with the excess CS emanating from the surface of test complexes, thus making them more compact. With γ-PGA coating, the extent of test complexes internalized and their transfection efficiency were evidently enhanced. Trypsin treatment induced a concentration-dependent decrease in internalization of the γ-PGA-coated complexes, suggesting a specific protein-mediated endocytosis. The endocytosis inhibition study indicates that the γ-glutamyl transpeptidase (GGT) present on cell membranes was responsible for the uptake of test complexes. The amine group in the N-terminal γ-glutamyl unit on γ-PGA played an essential role in the interaction with GGT. When entangled with CS, the free N-terminal γ-glutamyl unit of γ-PGA on test complexes was exposed and might thus be accommodated within the γ-glutamyl binding pocket of the membrane GGT. Above results suggest that the γ-PGA coating on CS/DNA complexes can significantly enhance their cellular uptake via a specific GGT-mediated pathway. Knowledge of the uptake mechanism is crucial for the development of an efficient vector for gene transfection.     

Foci of altered hepatocytes in a fox squirrel (Sciurus niger) with haemochromatosis

Numerous minute milky white foci were distributed throughout the dark brown liver in an adult male fox squirrel. Histologically, the hepatic focal lesions were composed of large eosinophilic granular hepatocytes, which were mostly positive for glutathione S-transferase mu antigen and proliferating cell nuclear antigen. Electron microscopy demonstrated an increased number of mitochondria. These features corresponded to those in the eosinophilic type of foci of altered hepatocytes. Berlin blue stain showed severe haemosiderin deposition in hepatocytes, except in the focal lesions. Since the fox squirrel is known to be liable to develop congenital porphyria, it is suggested that the hepatic anomalies described may be closely associated with the development of porphyria.     

In vivo cell lineage analysis during chemical hepatocarcinogenesis in rats using retroviral-mediated gene transfer: evidence for dedifferentiation of mature hepatocytes

Feeding adult rats with a diet containing 2-acetylaminofluorene (2-AAF) results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. Although oval cells may be facultative liver stem cells, the actual relationship between oval cells and liver cancer has not been clearly established in vivo. Our goal was to label hepatic cells in vivo using retroviral vectors and follow their fate during the early steps of chemically induced hepatocarcinogenesis. Oval cell proliferation was induced by continuous feeding with a carcinogenic diet containing 2-AAF. We used two different strategies to genetically label hepatic cells: (a) labeling of proliferating cells in rats fed 2-AAF by injecting recombinant retroviral vectors containing the beta-galactosidase gene either in a peripheral vein or in the common bile duct at the peak of oval cell proliferation and (b) prelabeling of hepatocytes by intravenously injecting recombinant vectors 1 day after partial hepatectomy and 1 week before subsequent administration of 2-AAF. Using the first strategy, transgene expression occurred in both oval cells and hepatocytes. Using the second strategy, we could selectively label, and hence study the fate of, differentiated hepatocytes. In the latter case, we observed clusters of beta-galactosidase-positive hepatocytes, some of them also expressing preneoplastic markers such as gamma-glutamyl transpeptidase as well as the placental form of glutathione-S-transferase. These results demonstrate that preneoplastic foci can originate from mature hepatocytes and are consistent with the hypothesis that dedifferentiation of mature hepatocytes may occur during the course of carcinogenic regimen.