Preneoplastic Foci Of Altered Hepatocytes Research Articles

Effects of sorafenib and cisplatin on preneoplastic foci of altered hepatocytes in fetal turkey liver.

Foci of altered hepatocytes (FAH) were induced in fetal turkey liver (FTL) by diethyl nitrosamine. FAH in FTL were resistant to iron overload similar to FAH in humans and rodents. The mitotic index was significantly higher in FAH (6.2 mitosis in 1000 hepatocytes) than in extrafocal liver tissue (1.8 mitosis in 1000 hepatocytes). The calculation of the net growth rate based on both cell proliferation (mitosis) and cell death (TUNEL positive) revealed a threefold growth advantage of the FAH over the surrounding liver. Two well established anti-tumor substances from different chemical classes, different modes of action and with different clinical use in the treatment of hepatocellular carcinoma (HCC) were used to study their effect on FAH. Sorafenib is the only approved drug for systemic pharmacological treatment of HCC; cisplatin has been used for many years in hepatic arterial infusion. Cisplatin had no clear effect on number of size of FAH, cell proliferation (mitosis) or cell loss (TUNEL positive). Sorafenib enhanced the development of FAH. Morphometric quantification revealed a sorafenib-induced 2-3-fold increase in number (FAH per cm2 and FAH per cm3), size and volume fraction of FAH. This unexpected finding was confirmed in two experiments. The effect was driven by an increased cell proliferation in the FAH, resulting in an increased, 5.4-fold growth advantage of FAH versus the surrounding liver in sorafenib-treated FTL. In this model, sorafenib has a promoting effect on preneoplastic FAH. This might be of relevance for the treatment of patients with long term survival perspective, e.g. in an adjuvant setting.

Dietary tomato powder inhibits alcohol-induced hepatic injury by suppressing cytochrome p450 2E1 induction in rodent models

Chronic and excessive alcohol consumption leads to the development of alcoholic liver disease (ALD) and greatly increases the risk of liver cancer. Induction of the cytochrome p450 2E1 (CYP2E1) enzyme by chronic and excessive alcohol intake is known to play a role in the pathogenesis of ALD. High intake of tomatoes, rich in the carotenoid lycopene, is associated with a decreased risk of chronic disease. We investigated the effects of whole tomato (tomato powder, TP), partial tomato (tomato extract, TE), and purified lycopene (LYC) against ALD development in rats. Of the three supplements, only TP reduced the severity of alcohol-induced steatosis, hepatic inflammatory foci, and CYP2E1 protein levels. TE had no effect on these outcomes and LYC greatly increased inflammatory foci in alcohol-fed rats. To further support the protective effect of TP against ALD, TP was supplemented in a carcinogen (diethylnitrosamine, DEN)-initiated alcohol-promoted mouse model. In addition to reduced steatosis and inflammatory foci, TP abolished the presence of preneoplastic foci of altered hepatocytes in DEN-injected mice fed alcohol. These reductions were associated with decreased hepatic CYP2E1 protein levels, restored levels of peroxisome proliferator-activated receptor-α and downstream gene expression, decreased inflammatory gene expression, and reduced endoplasmic reticulum stress markers. These data provide strong evidence for TP as an effective whole food prevention strategy against ALD.

Comparative Aspects of Rat and Human Hepatocellular Preneoplasia and Neoplasia

In a “comparative histomorphological review of rat and human hepatocellular proliferative lesions”, Thoolen and colleagues1 stated that “there are major similarities in the diagnostic features, growth patterns and behavior of both rat and human proliferative hepatocellular lesions and in the process of hepatocarcinogenesis”. I fully agree with this statement, but in view of its far-reaching consequences for the evaluation of rodent carcinogenesis bioassays and the early detection of precursors of human hepatocellular adenomas (HCA) and carcinomas (HCC) I draw your attention to several critical comparisons and conclusions concerning the significance of preneoplastic foci of altered hepatocytes (FAH), called “foci of cellular alteration” in the review.

Upregulation of the Insulin Receptor and Type I Insulin-Like Growth Factor Receptor Are Early Events in Hepatocarcinogenesis

The molecular mechanisms underlying the development of hepatocellular carcinoma (HCC) are not yet fully understood. Preneoplastic foci of altered hepatocytes regularly precede HCC in various species. The predominant earliest type of foci of altered hepatocytes, the glycogen storage focus (GSF), shows an excess of glycogen (glycogenosis) in the cytoplasm. During progression from GSF to HCC, the stored glycogen is gradually reduced, resulting in complete loss in basophilic HCC. We have previously shown that in N-nitrosomorpholine-induced hepatocarcinogenesis, insulin receptor substrate (IRS-1) is strongly expressed in GSF and reduced during progression to HCC, thus correlating with the glycogen content. In the present study, we observed increased levels of insulin receptor, IGF-I receptor (IGF-IR), IRS-2, and mitogen-activated kinase/extracellular regulated kinase-1 in GSF, following the same pattern of expression as IRS-1. We conclude that the abundance of IRS-1, IRS-2, and mitogen-activated kinase/extracellular regulated kinase-1 coincides with a concerted upregulation of both IR and IGF-IR induced by the hepatocarcinogen. Our data suggest that in early hepatocellular preneoplasia, the upregulation of IR elicits glycogenosis through IRS-1 and/or IRS-2, whereas the increased level of the IGF-IR may lead to the increased cell proliferation previously reported in GSF. Therefore, the concerted upregulation of both IR and IGF-IR may represent initial events in hepatocarcinogenesis.

The Ras inhibitor farnesylthiosalicyclic acid (FTS) prevents nodule formation and development of preneoplastic foci of altered hepatocytes in rats

Background Aberrant activation of oncogenes, such as Ras, likely contributes to the development of hepatocarcinoma (HCC). Aims/methods We evaluated in vivo the effect of intraperitoneal injections of the Ras inhibitor S-trans, trans-farnesylthiosalicyclic acid (FTS) on Ras activation and the development of preneoplastic liver lesions in rats receiving weekly diethylnitrosamine (DEN) injections for 16 weeks. Western blotting, quantitative PCR, immunohistochemistry, Tunel and caspase activity assays were used. Results FTS prevents liver nodule formation and reduces foci expressing the tumour marker GSTp. FTS abrogates DEN-induced Ras membrane activity, increases Tunel positive cells in transformed, GSTp-expressing hepatocytes, up-regulates caspase 3 and 8 activity, induces Fas, Fas ligand and JNK phosphorylation that occurs independently of TNFα and Trail. Cytochrome C release, Bax, Bcl2, Bcl-xl, Ki67 and nuclear cyclin D expression is not affected by FTS. Conclusions FTS inhibits Ras activation and prevents preneoplastic liver nodule development by inducing apoptosis in transformed hepatocytes through activation of the Fas/Fas ligand system. FTS might be new molecule for HCC treatment.

I-3 The Ras inhibitor farnesylthiosalicyclic acid (FTS) prevents nodule formation and development of preneoplastic foci of altered hepatocytes in rats

I-3 The Ras inhibitor farnesylthiosalicyclic acid (FTS) prevents nodule formation and development of preneoplastic foci of altered hepatocytes in rats

Protective effects of Ginkgo biloba against rat liver carcinogenesis

Ginkgo biloba (EGb) has been proposed as a promising candidate for cancer chemoprevention and has shown protective effects on the liver against chemically induced oxidative injury and fibrosis. The potential beneficial effects of EGb were investigated in two rat liver carcinogenesis bioassays induced by diethylnitrosamine (DEN). In a short-term study for anti-initiating screening, male Wistar rats were fed a basal diet or supplemented diet with 500 or 1000 ppm EGb and initiated 14 days later with a single dose of DEN (100 mg/kg i.p.). The respective groups were killed 24 h or 2 weeks after DEN-initiation. Liver samples were collected for the analysis of proliferating cell nuclear antigen (PCNA), transforming growth factor alpha (TGF-α), p53, apoptosis and induction of single hepatocytes and minifoci positive for the enzyme glutathione S-transferase P-form (GST-P). In a medium-term study for anti-promoting screening, the animals received a single dose of DEN (200 mg/kg i.p.) and, 2 weeks later, were fed a basal diet or supplemented diet with 500 or 1000 ppm EGb for 6 weeks. All animals underwent 70% partial hepatectomy (PH) at week 3 and killed at week 8. Liver samples were colleted to analyze development of preneoplastic foci of altered hepatocytes (FAH) expressing GST-P. In the short-term study, pretreatment of rats with 1000 ppm EGb significantly reduced the rates of cell proliferation, apoptosis and p53, TGF-α immunoreactivity and the number of GST-P-positive hepatocytes. In the medium-term study, EGb treatment during the post-initiation stage failed to reduce the development of DEN-induced GST-P-positive foci. Thus, EGb presented inhibitory actions during initiation but not promotion of rat liver carcinogenesis induced by DEN.

Investigating the Formation and Growth of α-Particle Radiation-Induced Foci of Altered Hepatocytes: A Model-Based Approach

The effect of alpha-particle radiation on the formation and increase in volume of preneoplastic liver lesions was investigated in an animal experiment. Mice were divided into four groups; two groups received different doses of the alpha-particle-labeled antibody (213)Bi-anti CD19 ((213)Bi-CD19), Thorotrast was administered to one group, and one group was left untreated. Hematoxylin and eosin-stained liver sections were evaluated for preneoplastic foci of altered hepatocytes 6, 12 and 17 months after treatment. The density and size distribution of focal transections were described by a mechanistic model for the formation and growth of foci of altered hepatocytes. The negative control and the (213)Bi-CD19 groups were combined to investigate the dose-response relationship for model parameters describing the formation and growth of foci of altered hepatocytes. Although (213)Bi-CD19 was given by single injection, the effect on formation of foci of altered hepatocytes lasted for the entire experiment. Likelihood-ratio tests comparing nested models showed that (213)Bi-CD19 increases the rates of both the formation and growth of foci of altered hepatocytes. Comparing the effects of Thorotrast with those of (213)Bi-CD19 revealed that Thorotrast had an effect similar to that of a low dose of (213)Bi-CD19, but the effect on focus formation was slightly smaller whereas the effect on focus growth was slightly higher for Thorotrast, in contrast to a low dose of (213)Bi-CD19.

Hepatocellular Neoplasms Induced by Low-Number Pancreatic Islet Transplants in Autoimmune Diabetic BB/Pfd Rats

It has been shown that combined high local hyperinsulinism and hyperglycemia after low-number islet transplantation into the livers of streptozotocin-diabetic rats lead to the development of hepatocellular neoplasms but a substantial cocarcinogenic effect of genotoxic streptozotocin could not be ruled out completely. Thus, we herein investigated this model in BB/Pfd rats (n = 805; nine experimental groups), which develop spontaneous autoimmune diabetes similar to human type 1 diabetes. After low-number islet transplantation (n = 450), the liver acini downstream of the islets show insulin-induced alterations: massive glycogen and/or fat accumulation, translocation of the insulin receptor, decrease in glucose-6-phosphatase activity, increase in expression of insulin-like growth factor (IGF)-I, IGF-II/mannose-6-phosphate receptor, insulin receptor substrate-1, Raf-1, and Mek-1, corresponding to clear cell preneoplastic foci of altered hepatocytes known from chemical hepatocarcinogenesis and identical to that in streptozotocin-diabetic Lewis rats. After 6 months, many altered liver acini progressed to other types of preneoplasias often accompanied by an overexpression of the glutathione-S transferase (placental form), IGF-I receptor, and transforming growth factor (TGF)-alpha. After 12 to 15 and 15 to 18 months, 52% and 100% of the animals showed one or multiple hepatocellular adenomas or hepatocellular carcinomas (HCCs), respectively. Conclusively, this study identifies combined hyperinsulinism and hyperglycemia as a carcinogenic mechanism for the development of HCCs in diabetic rats. Hepatocarcinogenesis is independent from additional genotoxic compounds (i.e., streptozotocin), but is primarily triggered by increased intracellular insulin signaling via pathways associated with cell growth and proliferation, such as the Ras-Raf-mitogen-activated protein kinase pathway and the IGF system, and secondarily involves other growth factors, such as TGF-alpha.

Hepatocellular neoplasms induced by low-number pancreatic islet transplants in autoimmune diabetic BB/Pfd rats

It has been shown that combined high local hyperinsulinism and hyperglycemia after low-number islet transplantation into the livers of streptozotocin-diabetic rats lead to the development of hepatocellular neoplasms but a substantial cocarcinogenic effect of genotoxic streptozotocin could not be ruled out completely. Thus, we herein investigated this model in BB/Pfd rats ( n = 805; nine experimental groups), which develop spontaneous autoimmune diabetes similar to human type 1 diabetes. After low-number islet transplantation ( n = 450), the liver acini downstream of the islets show insulin-induced alterations: massive glycogen and/or fat accumulation, translocation of the insulin receptor, decrease in glucose-6-phosphatase activity, increase in expression of insulin-like growth factor (IGF)-I, IGF-II/mannose-6-phosphate receptor, insulin receptor substrate-1, Raf-1, and Mek-1, corresponding to clear cell preneoplastic foci of altered hepatocytes known from chemical hepatocarcinogenesis and identical to that in streptozotocin-diabetic Lewis rats. After 6 months, many altered liver acini progressed to other types of preneoplasias often accompanied by an overexpression of the glutathione- S transferase (placental form), IGF-I receptor, and transforming growth factor (TGF)-α. After 12 to 15 and 15 to 18 months, 52% and 100% of the animals showed one or multiple hepatocellular adenomas or hepatocellular carcinomas (HCCs), respectively. Conclusively, this study identifies combined hyperinsulinism and hyperglycemia as a carcinogenic mechanism for the development of HCCs in diabetic rats. Hepatocarcinogenesis is independent from additional genotoxic compounds (i.e., streptozotocin), but is primarily triggered by increased intracellular insulin signaling via pathways associated with cell growth and proliferation, such as the Ras-Raf-mitogen-activated protein kinase pathway and the IGF system, and secondarily involves other growth factors, such as TGF-α. (Cancer Res 2006; 66(3): 1833-43)

Apolipoprotein A-IV mRNA Overexpression in Early Preneoplastic Hepatic Foci Induced by Low-number Pancreatic Islet Transplants in Streptozotocin-diabetic Rats

After low-number transplantation of islets of Langerhans into the liver of streptozotocin-diabetic rats, the hepatocytes in the acini, draining the blood from the islets, are exposed to a local hyperinsulinemia, whereas the remaining tissue is affected by hypoinsulinemia. In this model, insulin induces alterations that resemble preneoplastic foci of altered hepatocytes (FAH) and develop into hepatocellular tumors in later stages of carcinogenesis. In rodents, apolipoprotein A-IV (A-IV) is synthesized in the small intestine and the liver. Whereas intestinal production is mainly influenced by lipid intake and chylomicrone formation, little is known about mechanisms regulating hepatic A-IV synthesis. As it is known that insulin modulates lipoprotein metabolism in different ways, we investigated the effect of insulin on hepatocytic A-IV mRNA expression in this model. After Laser microdissection of FAH and quantitative RT-PCR (LightCycler), we found a 3.2 to 7.4-fold increase of A-IV mRNA in the FAH. To the best of our knowledge, these results represent the first data of insulin-stimulated A-IV mRNA overexpression in rat hepatocytes in vivo, and are in line with previously reported results of experiments with cultured hepatocytes. It remains to be elucidated whether A-IV mRNA overexpression is only an epiphenomenon of insulin action or is relevant for hepatocarcinogenesis in this model.

Prevalidation of a rat liver foci bioassay (RLFB) based on results from 1600 rats: a study report.

A rat liver foci bioassay (RLFB) based on an initiation-promotion protocol employing preneoplastic foci of altered hepatocytes (FAH) as an endpoint, was prevalidated in 5 different laboratories. FAH were identified by immunohistochemical demonstration of glutathione-S-transferase (placental form, GSTP) and by staining with hematoxilin/eosin (H&E), and their area fraction was quantified morphometrically. The four model hepatocarcinogens N-nitrosomorpholine, 2-acetylaminofluoren, phenobarbital, and clofibrate were selected according to characteristic differences in their presumed mode of action, and tested in a total of 1,600 male and female rats at 2 different dose levels. The chemicals were found to differ characteristically in their potency and dose-response relationship to induce FAH when given alone or when administered following initiation with diethylnitrosamine. The interlaboratory variation was small for results obtained with the GSTP-stain and somewhat larger with respect to H&E. The assessment of the carcinogenic potential of the four chemicals by the different laboratories was in the same range and the nature of their dose-response relationships did not differ essentially between laboratories. Our results suggest that this RLFB is a sensitive bioassay, providing potentially valuable information for risk assessment including the classification of carcinogenic chemicals according to their mode of action.

Significance of hepatic preneoplasia in risk identification and early detection of neoplasia.

Among the different types of liver tumor, hepatocellular neoplasms predominate by far in both animals and man. Consequently, preneoplastic foci of altered hepatocytes (FAH), preceding both hepatocellular adenomas and carcinomas, represent the most prevalent form of hepatic preneoplasia observed in animals for a long time, and identified in human chronic liver diseases associated with, or predisposing to, hepatocellular carcinomas more recently. Morphological, microbiochemical, and molecular biological approaches in situ revealed striking similarities in specific changes of the cellular phenotype of preneoplastic FAH developing in experimental and human hepatocarcinogenesis, irrespective of whether this was elicited by chemicals, hormones, viruses or radiation. The advantage of using FAH for risk identification (aiming at primary cancer prevention) in long-term and medium-term carcinogenesis bioassays has been well documented, but quantitative morphometric approaches appear to be indispensable for an appropriate evaluation of both bioassays. The detection of phenotypically similar FAH in various animal models and in humans prone to develop or bearing hepatocellular carcinomas favors the extrapolation from data obtained in animals to humans. Moreover, the recently reported frequent finding of FAH in fine-needle biopsies of patients suffering from chronic liver diseases opens new perspectives for secondary prevention of human hepatocellular carcinoma.

Development of hepatocellular adenomas and carcinomas associated with fibrosis in C57BL/6J male mice given a choline-deficient, L-amino acid-defined diet.

Development of hepatocellular carcinomas in rats caused by a choline‐deficient, L‐amino acid defined (CDAA) diet, usually associated with fatty liver, fibrosis, cirrhosis and oxidative DNA damage, has been recognized as a useful model of hepatocarcinogenesis caused by endogenous factors. In the present study, in order to further explore involved factors and genes, we established an equivalent model in spontaneous liver tumor‐resistant C57BL/6J mice. Six‐week‐old males and females were continuously fed the CDAA diet and histological liver lesions and oxidative DNA damage due to 8‐hydroxydeoxyguanosine (8‐OHdG) were examined after 22, 65 and 84 weeks. In male mice, fatty change and fibrosis were evident at 22 weeks, and preneoplastic foci of altered hepatocytes were seen at an incidence of 8/8 (100%) and a multiplicity of 6.6±4.0 per mouse at 65 weeks. Hepatocellular adenomas and carcinomas developed at incidences of 16/24 (66.7%) and 5/ 24 (20.8%), and multiplicities of 1.421±1.32 and 0.29±0.62, respectively, at 84 weeks. The female mice exhibited resistance to development of these lesions. The CDAA diet also increased 8‐OHdG levels in male but not female mice. These results indicate that a CDAA diet causes hepatocellular preneoplastic foci, adenomas and carcinomas associated with fibrosis and oxidative DNA damage in mice, as in rats, providing a hepatocarcinogenesis model caused by endogenous factors in mice.

Expression of MUC1, Thomsen-Friedenreich antigen, Tn, sialosyl-Tn, and alpha2,6-linked sialic acid in hepatocellular carcinomas and preneoplastic hepatocellular lesions.

The expression of epithelial mucins and Thomsen-Friedenreich-related antigens in preneoplastic and neoplastic hepatocellular lesions was systematically investigated using an in situ immunohistochemical staining approach. MUC1, MUC2, TF, sialosyl-TF, Tn, sialosyl-Tn, alpha2,3-linked sialic acid, and alpha2,6-linked sialic acid were examined in normal and cirrhotic human liver and in human hepatocellular carcinomas (HCCs) and cholangiocarcinomas. Normal hepatocytes and preneoplastic foci of altered hepatocytes did not express MUC1, MUC2, TF, Tn, s-Tn, or alpha2,6-linked sialic acid. In contrast, HCCs showed positive reactions for MUC1, TF, Tn, s-Tn, and alpha2,6-linked sialic acid. MUC2 was absent in normal biliary epithelial cells, but present in cholangiocarcinomas. The staining of MUC1, or s-Tn and alpha2,6-linked sialic acid in human normal liver tissues and various liver diseases did not change after specific treatments such as periodate oxidation or saponification, indicating that their expression in HCC does not result from incomplete glycosylation or low O-acetylation, respectively. MUC1, TF, Tn, s-Tn, and alpha2,6-linked sialic acid may be useful as indicators of progression of HCC in tissue sections, and perhaps also as targets for diagnostic and therapeutic approaches in vivo.

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.

Enhancement and phenotypic modulation of N-nitrosomorpholine-induced hepatocarcinogenesis by dehydroepiandrosterone

Hepatocarcinogenesis was induced in male and female rats by continuous administration of the adrenal steroid dehydroepiandrosterone (DHEA; 0.6% in the diet) with and without previous treatment with N-nitrosomorpholine (NNM; 120 mg/l drinking water for 7 weeks). DHEA treatment alone resulted in hepatocellular adenomas (HCA) and carcinomas (HCC) after 72–84 weeks, the incidence of both benign and malignant neoplasms being higher in females than in males. After DHEA administration for up to 32 weeks subsequent to NNM, the incidence of HCA and HCC was significantly higher (HCA, 42%; HCC, 42%) than after NNM alone (HCA, 33%; HCC, 28%). While total tumor incidence was similar in male (63%) and female (60%) rats after NNM treatment alone, it was higher in females (87%) than in males (80%) after NNM/DHEA treatment. The difference between the genders was mainly due to the higher incidence of HCC in females. Morphometric analysis of preneoplastic foci of altered hepatocytes (FAH) yielded that DHEA treatment did not increase the average total number of FAH induced by NNM, but caused a modulation of the phenotype of FAH from the glycogenotic/basophilic to the amphophilic cell lineage. The results confirm that DHEA acts as a hepatocarcinogen and show for the first time that it enhances NNM-induced hepatocarcinogenesis in rats.

Early bioenergetic changes in hepatocarcinogenesis: preneoplastic phenotypes mimic responses to insulin and thyroid hormone.

Biochemical and molecular biological approaches in situ have provided compelling evidence for early bioenergetic changes in hepatocarcinogenesis. Hepatocellular neoplasms regularly develop from preneoplastic foci of altered hepatocytes, irrespective of whether they are caused by chemicals, radiation, viruses, or transgenic oncogenes. Two striking early metabolic aberrations were discovered: (1) a focal excessive storage of glycogen (glycogenosis) leading via various intermediate stages to neoplasms, the malignant phenotype of which is poor in glycogen but rich in ribosomes (basophilic), and (2) an accumulation of mitochondria in so-called oncocytes and amphophilic cells, giving rise to well-differentiated neoplasms. The metabolic pattern of human and experimentally induced focal hepatic glycogenosis mimics the phenotype of hepatocytes exposed to insulin. The conversion of the highly differentiated glycogenotic hepatocytes to the poorly differentiated cancer cells is usually associated with a reduction in gluconeogenesis, an activation of the pentose phosphate pathway and glycolysis, and an ever increasing cell proliferation. The metabolic pattern of preneoplastic amphophilic cell populations has only been studied to a limited extent. The few available data suggest that thyromimetic effects of peroxisomal proliferators and hepadnaviral infection may be responsible for the emergence of the amphophilic cell lineage of hepatocarcinogenesis. The actions of both insulin and thyroid hormone are mediated by intracellular signal transduction. It is, thus, conceivable that the early changes in energy metabolism during hepatocarcinogenesis are the consequence of alterations in the complex network of signal transduction pathways, which may be caused by genetic as well as epigenetic primary lesions, and elicit adaptive metabolic changes eventually resulting in the malignant neoplastic phenotype.

Rapid induction of preneoplastic liver foci in embryonal turkey liver by diethylnitrosamine.

Diethylnitrosamine (DEN) was injected into fertilized turkey eggs. After incubation for 24 days, the livers of the embryos were removed and investigated by means of histological and enzyme-histochemical methods. Doses of 2 and 5 mg DEN per egg had no effect on mortality and embryo weight but induced various types of foci of altered hepatocytes. In addition, a replacement of the trabecular structure of the liver by a tubular arrangement of the hepatocytes and markedly enlarged hepatocytes were found. The diethylnitrosamine-induced foci of altered hepatocytes were very similar to the preneoplastic lesions that occur in the liver of mammals during hepatocarcinogenesis and are regarded as early indicators of carcinogenicity. The presented in ovo model is a simple (1 dose), rapid (24 days), and inexpensive (no animal housing) approach for the induction of foci of altered hepatocytes. The sensitivity of this carcinogenesis bioassay appears to be comparable to that of lifetime studies in rodents. It is proposed that the induction of preneoplastic foci of altered hepatocytes in ovo is a valuable screening model for hepatocarcinogenic effects.

Dose dependence of diethylnitrosamine-induced nuclear enlargement in embryonal turkey liver.

Avian embryos (turkey) were exposed to diethylnitrosamine in ovo. On the first day of incubation doses of 0.5-5.0 mg/egg were injected into the white of the fertilized egg. The experiment was terminated 4 days before hatching. Livers were removed and prepared for subsequent histological examination. In haematoxylin and eosin stained sections the areas of hepatocyte nuclear profiles were measured by semi-automatic image analysis. In liver samples of diethylnitrosamine-exposed embryos hepatocyte nuclei of more than twice the size of normal hepatocyte nuclei were found. The incidence of the enlarged nuclei was clearly dose dependent. An increase in the size of hepatocyte nuclei was observed after low doses of diethylnitrosamine that did not induce common signs of non-specific toxic effects, e.g. cell death, fat vacuoles or loss of glycogen. The slope of the dose-response curve was rather steep. A 10-fold increase in the dose of the carcinogen resulted in a 100-fold increase in the incidence of enlarged hepatocyte nuclei. In combination with preneoplastic foci of altered hepatocytes, the quantification of nuclear enlargement can provide a valuable complementary parameter for the evaluation of carcinogen-induced effects in ovo.