Mixed Cell Foci Research Articles

Low dose assessment of the carcinogenicity of furan in male F344/N Nctr rats in a 2-year gavage study

Furan is a volatile organic chemical that is a contaminant in many common foods. Furan is hepatocarcinogenic in mice and rats; however, the risk to humans from dietary exposure to furan cannot be estimated accurately because the lowest tested dose of furan in a 2-year bioassay in rats gave nearly a 100% incidence of cholangiocarcinoma. To provide bioassay data that can be used in preparing risk assessments, the carcinogenicity of furan was determined in male F344/N Nctr rats administered 0, 0.02, 0.044, 0.092, 0.2, 0.44, 0.92, and 2 mg furan/kg body weight (BW) by gavage 5 days/week for 2 years. Exposure to furan was associated with the development of malignant mesothelioma on membranes surrounding the epididymis and on the testicular tunics, with the increase being significant at 2 mg furan/kg BW. There was also a dose-related increase in the incidence of mononuclear cell leukemia, with the increase in incidence being significant at 0.092, 0.2, 0.92, and 2 mg furan/kg BW. Dose-related non-neoplastic liver lesions included cholangiofibrosis, mixed cell foci, basophilic foci, biliary tract hyperplasia, oval cell hyperplasia, regenerative hyperplasia, and cytoplasmic vacuolization. The most sensitive non-neoplastic lesion was cholangiofibrosis, the frequency of which increased significantly at 0.2 mg furan/kg BW.

Specific alterations of carbohydrate metabolism are associated with hepatocarcinogenesis in mitochondrially impaired mice

Friedreich's ataxia is an inherited neurodegenerative disease caused by the reduced expression of the mitochondrially active protein frataxin. We have previously shown that mice with a hepatocyte-specific frataxin knockout (AlbFxn(-/-)) develop multiple hepatic tumors in later life. In the present study, hepatic carbohydrate metabolism in AlbFxn(-/-) mice at an early and late life stage was analyzed. In young (5-week-old) AlbFxn(-/-) mice hepatic ATP, glucose-6-phosphate and glycogen levels were found to be reduced by ∼74, 80 and 88%, respectively, when compared with control animals. This pronounced ATP, G6P and glycogen depletion in the livers of young mice reverted in older animals: while half of the mice die before 30 weeks of age, the other half reaches 17 months of age and exhibits glycogen, G6P and ATP levels similar to those in age-matched controls. A key event in this respect seems to be the up-regulation of GLUT1, the predominant glucose transporter in fetal liver parenchyma, which became evident in AlbFxn(-/-) mice being 5-12 weeks of age. The most significant histological findings in animals being 17 or 22 months of age were the appearance of multiple clear cell, mixed cell and basophilic foci throughout the liver parenchyma as well as the development of hepatocellular adenomas and carcinomas. The hepatocarcinogenic process in AlbFxn(-/-) mice shows remarkable differences regarding carbohydrate metabolism alterations when compared with all other chemically and virally driven liver cancer models described up to now.

Changes in the antioxidant defense and hepatic drug metabolizing enzyme and isoenzyme levels, 8-hydroxydeoxyguanosine formation and expressions of c-raf.1 and insulin-like growth factor II genes during the stages of development of hepatocellular carcinoma in rats

This is an extensive study in a defined initiation-promotion hepatocellular carcinoma model of hepatocarcinogenesis (in rats) in which many important marker enzymes and isoenzymes and 8-hydroxydeoxyguanosine formation have been studied together with two very important cellular proliferating genes, insulin-like growth factor II and c-raf.1, known for their role in hepatocellular cancer development. Experiments were carried out on hepatic tissues of male Sprague-Dawley rats. Variations in different enzyme/isoenzyme activities/contents/expression pattern and 8-hydroxydeoxyguanosine-positive cells were studied. Insulin-like growth factor II and c-raf.1 gene expressions were monitored. A direct shift with increase in size and numbers of lesions was found to occur in different experimental groups. In this study, glutathione peroxidase (1.14 and 1.46-fold) and reduced triphosphopyridine nucleotide (TPNH)-cytochrome-c-reductase (1.94 and 2.94-fold) activities, cytochrome b5 (1.57 and 3.28-fold) and P-450 contents (1.45 and 1.22-fold), glutathione content (1.27 and 1.45-fold) and superoxide dismutase and catalase (1.16 and 1.39-fold) activities in group A animals were found to be lower than those in initiation and promotion studies, respectively. 8-Hydroxydeoxyguanosine-positive nuclei count showed that oxidative damage of nuclear DNA enhanced with the progress of the disease. The insulin-like growth factor II expression was found to be predominant in hepatocellular carcinoma and in early preneoplastic lesions. Unlike insulin-like growth factor II, c-raf.1 expression was located in the late basophilic lesions associated with hepatocellular carcinoma. During the various stages of the development of hepatocellular carcinoma, the enzymes played a significant role in metabolizing carcinogens and thereby scavenging various toxic metabolites or free radicals produced. A sequence of cellular changes starting from the appearance of glycogen storage foci to basophilic foci leading to hepatocellular carcinoma via mixed cell foci varied the activity/content or expression pattern of the enzymes and isoenzymes and in 8-hydroxydeoxyguanosine formation. It has been established that c-raf.1-induced signaling pathways activated by insulin-like growth factor II is implicated in the late stage of development of cancer.

Decreased density of beta1-adrenergic receptors in preneoplastic and neoplastic liver lesions of F344 rats.

There is some evidence that rodent hepatocarcinogenesis is accompanied by changes in the adrenergic responsiveness of liver cells to catecholamines. In this study, immunohistochemical expression of beta1-adrenergic receptors (beta1-ARs) has been examined in spontaneous and chemically induced preneoplastic and neoplastic liver lesions of female and male Fischer 344 rats. An antibody specific for beta1-AR subtype was used. The study was carried out on archival formalin-fixed and paraffin-embedded livers from rats used in a previous study of hepatocarcinogenesis. One control group given distilled water by gavage, and two experimental groups, one initiated with a single dose of diethylnitrosamine (DEN) and one initiated with DEN and continuously treated with phenobarbital (PB) were examined. Rats were sacrificed after 2, 4, 8 and 21 months of experimentation. All types of liver putative preneoplastic lesions examined (basophilic, glycogen-retaining, or mixed cell foci) show a lower density of beta1-ARs than the surrounding normal liver parenchyma, either in control and in DEN-treated or DEN+PB-treated rats. No immunostaining is detectable in several altered cell foci. Hepatocellular adenomas and hepatocellular carcinomas also show a very low density of beta1-ARs, extensive areas completely devoid of beta1-ARs being mingled with areas showing a weak immunostaining.

Hepatomegaly in transgenic mice expressing the homeobox gene Cux-1.

Cux-1 is a member of a family of homeobox genes structurally related to Drosophila Cut. Mammalian Cut proteins function as transcriptional repressors of genes specifying terminal differentiation in multiple cell lineages. In addition, mammalian Cut proteins serve as cell-cycle-dependent transcriptional factors in proliferating cells, where they function to repress expression of the cyclin kinase inhibitors p21 and p27. Previously we showed that transgenic mice expressing Cux-1 under control of the CMV immediate early gene promoter develop multiorgan hyperplasia. Here we show that mice constitutively expressing Cux-1 exhibit hepatomegaly correlating with an increase in cell proliferation. In addition, the increase in Cux-1 expression in transgenic livers was associated with a decrease in p21, but not p27, expression. Within transgenic livers, Cux-1 was ectopically expressed in a population of small cells, but not in mature hepatocytes, and many of these small cells expressed markers of proliferation. Transgenic livers showed an increase in alpha-smooth muscle actin, indicating activation of hepatic stellate cells, and an increase in cells expressing chromogranin-A, a marker for hepatocyte precursor cells. Morphological analysis of transgenic livers revealed inflammation, hepatocyte swelling, mixed cell foci, and biliary cell hyperplasia. These results suggest that increased expression of Cux-1 may play a role in the activation of hepatic stem cells, possibly through the repression of the cyclin kinase inhibitor p21.

Relevance of hepatic preneoplasia for human hepatocarcinogenesis.

Different lesions have been suggested to represent preneoplastic conditions in human liver. They include liver cell dysplasia, separated in large-cell change (LCC) and small-cell change (SCC), adenomatoid hyperplasia, and the more recently identified foci of altered hepatocytes (FAH) and nodules of altered hepatocytes (NAH). FAH have been demonstrated to represent preneoplastic lesions in various animal models of hepatocarcinogenesis. To demonstrate prevalence and significance of FAH in the human liver, the cellular composition, size distribution, and proliferation kinetics of these lesions were studied in 163 explanted and resected human livers with or without hepatocellular carcinoma (HCC). FAH including glycogen-storing foci (GSF), mixed cell foci (MCF), and basophilic cell foci were found in 84 of 111 cirrhotic livers, demonstrating higher incidences in cases with than without HCC. MCF, predominant in cirrhotic livers of the high-risk group, were more proliferative, larger and more often involved in formation of NAH than GSF. The results suggest that the FAH are preneoplastic lesions, MCF being more advanced than GSP. We also investigated the relationship of FAH to liver cell dysplasia. Occurrence of SCC, rather than that of LCC, confers FAH an increased proliferation activity and higher risk to nodular transformation, and, hence, should be considered a precancerous condition. Histological detection of FAH and SCC through needle-aspiration liver biopsy can be used for monitoring HCC development in high-risk populations, such as HBV carriers with chronic hepatitis and/or cirrhosis.

Two-Year Toxicity and Carcinogenicity Study of Methyleugenol in F344/N Rats and B6C3F1Mice

Methyleugenol (MEG) was tested for toxicity/carcinogenicity in a 2-yr carcinogenesis bioassay because of its widespread use in a variety of foods, beverages, and cosmetics as well as its structural resemblance to the known carcinogen safrole. F344/N rats and B6C3F(1) mice (50 animals/sex/dose group) were given MEG suspended in 0.5% methylcellulose by gavage at doses of 37, 75, or 150 mg/kg/day for 2 yr. Control groups (60 rats/sex and 50 mice/sex) received only the vehicle. A stop-exposure group of 60 rats/sex received 300 mg/kg/day by gavage for 53 weeks followed by the vehicle only for the remaining 52 weeks of the study. A special study group (10 animals/sex/species/dose group) were used for toxicokinetic studies. All male rats given 150 and 300 mg/kg/day died before the end of the study; survival of female rats given 150 mg/kg/day and all treated female mice was decreased. Mean body weights of treated male and female rats and mice were decreased when compared to control. Area under the curve results indicated that greater than dose proportional increases in plasma MEG occurred for male 150 and 300 mg/kg/day group rats (6 and 12 month) and male 150 mg/kg/day mice (12 month). Target organs included the liver, glandular stomach, forestomach (female rats) and kidney, mammary gland, and subcutaneous tissue (male rats). Liver neoplasms occurred in all dose groups of rats and mice and included hepatoadenoma, hepatocarcinoma, hepatocholangioma (rats only), hepatocholangiocarcinoma, and hepatoblastoma (mice only). Nonneoplastic liver lesions included eosinophilic and mixed cell foci (rats only), hypertrophy, oval cell hyperplasia, cystic degeneration (rats only), and bile duct hyperplasia. Mice also exhibited necrosis, hematopoietic cell proliferation, and hemosiderin pigmentation. Glandular stomach lesions in rats and mice included benign and malignant neuroendocrine tumors, neuroendocrine cell hyperplasia, and atrophy and in mice included glandular ectasia/chronic active inflammation. In female rats, the forestomach showed a positive trend in the incidences of squamous cell papilloma or carcinoma (combined). Male rats also exhibited kidney (renal tubule hyperplasia, nephropathy, and adenomacarcinoma), mammary gland (fibroadenoma), and subcutaneous tissue (fibroma, fibrosarcoma) lesions. Male rats also exhibited malignant mesotheliomas and splenic fibrosis. These data demonstrate that MEG is a multisite, multispecies carcinogen.

Insulin receptor substrate-1 is over-expressed in glycogenotic but not in amphophilic preneoplastic hepatic foci induced in rats by N-nitrosomorpholine and dehydroepiandrosterone

Insulin receptor substrate-1 (IRS-1) is over-expressed in preneoplastic glycogenotic hepatic foci (GSF) and is gradually down-regulated during progression of these lesions, via mixed cell foci (MCF), to the basophilic neoplastic phenotype. The aim of the present study was to investigate the effect of dehydroepiandrosterone (DHEA), a weak hepatocarcinogen and tumour enhancer, on IRS-1 expression. Hepatocellular lesions were induced by N-nitrosomorpholine followed by DHEA. Under these conditions, many glycogen-poor amphophilic (APF) and intermediate cell foci (ICF) appear, in addition to GSF and MCF. IRS-1 was over-expressed in 215 out of 295 GSF, in 50 out of 53 MCF and in a glycogen-rich mixed cell adenoma. IRS-1 expression was not shown in 147 APF, 51 ICF and 5 amphophilic hepatocellular adenomas, and 3 out of 5 hepatocellular carcinomas showed a weak IRS-1 expression. The results suggest a close association of IRS-1 over-expression with the glycogenotic hepatocellular phenotype. The modulation and enhancement of tumour progression by DHEA is associated with a shift from glycogenosis to amphophilia and basophilia, and a down-regulation of IRS-1 expression.

Xenomorphic hepatocellular precursors and neoplastic progression of tigroid cell foci induced in rats with low doses of N-nitrosomorpholine.

Tigroid cell foci (TCF) are a well-defined entity induced in rat liver by chemical carcinogens, their significance for hepatocarcinogenesis being controversial. Using cytomorphological, cytochemical and morphometric approaches, we studied the evolution and fate of TCF sequentially from 7 to 110 weeks in groups of 50 male Sprague-Dawley rats, which remained untreated or received N-nitrosomorpholine (NNM) orally at concentrations of 3 and 1 mg/kg body wt/day for 7 and up to 75 weeks, respectively. An increased incidence of hepatocellular neoplasms developed in exposed animals compared with controls, which was significant for adenomas at both dose levels, and for carcinomas (HCC) after the longer exposure to the lower dose level (P < 0.0001). TCF appeared frequently in addition to other types of proliferative foci of altered hepatocytes (FAH) including clear/acidophilic and mixed cell foci (MCF) in NNM-treated and rarely in untreated rats. Striking similarities in the cellular phenotypes of TCF and many hepatocellular neoplasms indicated the potential of TCF for progression to both adenomas and carcinomas. TCF emerged from xenomorphic cell foci (XCF), which consisted of hypertrophied hepatocytes typically presenting an enlarged nucleus, abundant glycogen, smooth and rough endoplasmic reticulum, altered activities of several enzymes of carbohydrate metabolism and an increased cell proliferation (P < 0.001) compared with the extrafocal parenchyma. TCF shared many features with XCF, but their basophilia and proliferative activity was higher. The number of FAH appearing at the two dose levels of NNM was similar but the average size of TCF and MCF was frequently higher at late time points in the group developing a significantly higher incidence of HCC, which suggests a pronounced acceleration of neoplastic conversion in established preneoplastic cell populations rather than the induction of additional FAH by sustained effects of low doses of carcinogens.

Focal hepatic glycogenosis

Foci of altered hepatocytes (FAH) including clear cell foci excessively storing glycogen (focal hepatic glycogenosis) are well known as preneoplastic lesions in animal models of hepatocarcinogenesis induced by chemical, physical or viral agents. The occurrence of similar lesions has been studied in a series of 67 explanted and 2 resected human livers using histological and histochemical approaches. A high incidence of FAH was found in the liver of patients suffering from hepatocellular carcinoma(HCC, 14/14) and liver cirrhosis (21/42). FAH were also detected in one patient each with inborn hepatic glycogenosis type 1a, and cholangiocellular carcinoma. Two patients with focal nodular hyperplasia had FAH-like enzymatic changes within these lesions. No FAH were found in 5 donor livers. FAH excessively storing glycogen including clear and mixed cell foci predominated in most cases with these lesions. The focal hepatic glycogenosis was associated with a significantly increased cell proliferation compared to the extrafocal parenchyma, and with alterations in the activity of various enzymes. In the 175 FAH studied by enzyme histochemistry, two enzymes involved in glycogen breakdown, namely glycogen phosphorylase and glucose-6-phosphatase, showed the most consistent changes, being reduced in 98% and 95%, respectively. In addition, the activities of adenosine triphosphatase and gamma-glutamyltransferase were reduced in 46% and 53% of FAH, respectively. Inconsistent changes were observed in FAH concerning a number of other enzymes. The 14 HCCs investigated histochemically often contained clear cell populations rich in glycogen in well differentiated portions, but were poor in glycogen in moderately and poorly differentiated tumors or tumor components. There were some similarities in the enzyme histochemical pattern of HCC and FAH but also important differences were evident. In contrast to FAH, all HCCs (except one carcinoma of the fibrolamellar type) showed an increase in the activity of the mitochondrial glycerol-3-phosphate dehydrogenase, and 50% of the cases had increased glucose-6-phosphate dehydrogenase activity. The activities of glucose-6-phosphatase and gamma-glutamyltransferase usually showed a reactivation, or even an increase compared to the extrafocal parenchyma, in moderately and poorly differentiated HCCs. Our results indicate that the focal hepatic glycogenosis is a putative preneoplastic lesion in human beings similar to laboratory animals. The focal hepatic glycogenosis appears to be a frequent initial step in neoplastic transformation of hepatocytes, a process associated with a fundamental shift in energy metabolism.

Transforming Growth Factor-α in Carcinogen- Induced F344 Rat Hepatic Foci

Transforming growth factor-α (TGFα) is a positive growth regulator in epithelial cells, including hepatocytes. Overexpression of TGFα has been associated with increased growth and malignancy of end-stage cancers in humans and rodents. The overall aim of this study was to characterize TGFα staining in diethylnitrosamine-induced hepatic foci from male F344 rats with the hematoxylin and eosin (H and E) histological phenotype. The association between the individual focal DNA synthesis labeling index and the presence of TGFα was also examined. Hepatic foci were identified as eosinophilic, basophilic, clear cell, or mixed cell. Of these foci, 37.5% labeled positive for TGFα. There were distinct differences in the pattern of TGFα labeling between the different H and E histological phenotypes. Intense, uniform TGFα labeling was observed in eosinophilic foci. Basophilic foci labeled for TGFα diffusely uniform throughout the cytoplasm. In clear-cell foci, TGFα labeling occurred primarily along the periphery of the cell membrane. In mixed-cell foci, labeling occurred both along the periphery and diffusely throughout the cytoplasm. On those slides stained, glutathione-S-transferase (placental; GSTP) was detected in almost all eosinophilic and mixed-cell foci, whereas approximately half of the basophilic and clear-cell foci stained for GSTP. The presence of GSTP in a focus was not always associated with the presence of increased TGFα protein. All rat hepatic adenomas and the one carcinoma labeled positive for TGFα. Increased levels of TGFα protein were associated with increased DNA synthesis labeling index. The number of TGFα-positive foci with the highest DNA synthesis labeling indices were statistically higher than those with lower levels of DNA synthesis labeling. Although characteristic staining patterns for TGFα were associated with specific histological subtype, the role that TGFα plays in the progression of focal lesions to neoplasia requires further definition. High levels of TGFα protein appear to be acquired sometime during the hepatocarcinogenic process. It may be that early lesions that acquire high levels of TGFα are the ones to develop into hepatocellular carcinoma (e.g., hepatocellular carcinoma is determined very early in the carcinogenic process). It is apparent that further work is needed to delineate the role of TGFα in both rodent and human hepatocarcinogenesis.

Triprolidine: 104-Week Feeding Study in Rats

Triprolidine: 104-Week Feeding Study in Rats. Greenman, D. L., Sheldon, W., Schieferstein, G., Allen, R., and Allaben, W. T. (1995). Fundam. Appl. Toxicol. 27, 223-231. The antihistamine, triprolidine hydrochloride, was fed at dietary concentrations of 0, 250, 1000, or 2000 ppm (as the free base) to groups of 60 Fischer 344 (F344) rats of each sex for up to 2 years to evaluate its potential carcinogenicity. Up to 12 per sex from each group were killed at 65 weeks, and hematology, clinical chemistry, and histopathology were evaluated. A complete histopathological evaluation was performed on all other animals; survivors were killed at 2 years. Survival was significantly extended in triprolidine-treated males and females, particularly at the high dose. At the close of the study high-dose males and females had gained significantly less body weight than controls. Among rats killed at 65 weeks females in the mid- and high-dose groups weighed significantly less than controls, but weights of control and dosed males were not significantly different. The incidences of numerous lesions tended to decrease with increasing triprolidine dose. In females, clitoral gland adenomas, thyroid c-cell hyperplasia and neoplasia, mammary gland hyperplasia and fibroadenomas, and uterine stromal polyps, and in males, anterior pituitary gland adenomas, preputial gland neoplasia, thyroid c-cell hyperplasia, pancreatic islet neoplasia, mononuclear cell leukemia, and the combination of lymphocytic, histiocytic, and undifferentiated cell malignant lymphomas and mononuclear leukemia, all exhibited negative dose trends. Cytoplasmic alterations of the parotid gland and numerous liver lesions tended to be more frequent in treated than in control animals. Liver lesions that exhibited positive dose trends include chronic inflammation and centrilobular fatty change in both sexes, mixed cell foci, and the combination of mixed cell foci and eosinophilic foci in females, and in males, basophilic foci and eosinophilic foci. Triprolidine was not carcinogenic in F344 rats.

Triprolidine: 104-Week Feeding Study in Rats

The antihistamine, triprolidine hydrochloride, was fed at dietary concentrations of 0, 250, 1000, or 2000 ppm (as the free base) to groups of 60 Fischer 344 (F344) rats of each sex for up to 2 years to evaluate its potential carcinogenicity. Up to 12 per sex from each group were killed at 65 weeks, and hematology, clinical chemistry, and histopathology were evaluated. A complete histopatho-logical evaluation was performed on all other animals; survivors were killed at 2 years. Survival was significantly extended in tri-prolidine-treated males and females, particularly at the high dose. At the close of the study high-dose males and females had gained significantly less body weight than controls. Among rats killed at 65 weeks females in the mid- and high-dose groups weighed significantly less than controls, but weights of control and dosed males were not significantly different. The incidences of numerous lesions tended to decrease with increasing triprolidine dose. In females, clitoral gland adenomas, thyroid c-cell hyperplasia and neoplasia, mammary gland hyperplasia and fibroadenomas, and uterine stromal polyps, and in males, anterior pituitary gland adenomas, preputial gland neoplasia, thyroid c-cell hyperplasia, pancreatic islet neoplasia, mononuclear cell leukemia, and the combination of lymphocytic, histiocytic, and undifferentiated cell malignant lymphomas and mononuclear leukemia, all exhibited negative dose trends. Cytoplasmic alterations of the parotid gland and numerous liver lesions tended to be more frequent in treated than in control animals. Liver lesions that exhibited positive dose trends include chronic inflammation and centrilobular fatty change in both sexes, mixed cell foci, and the combination of mixed cell foci and eosinophilic foci in females, and in males, basophilic foci and eosinophilic foci. Triprolidine was not carcinogenic in F344 rats.

OVEREXPRESSION OF GLUCOSE-6-PHOSPHATE-DEHYDROGENASE IN RAT HEPATIC PRENEOPLASIA AND NEOPLASIA

Abundance of messenger RNA (mRNA) and activity of glucose-6-phosphate dehydrogenase (G6PDH), the rate limiting enzyme of the oxidative pentose phosphate pathway, were studied in preneoplastic foci of altered hepatocytes (FAH) and hepatocellular carcinomas (HCC) induced in rats by limited oral exposure to N-nitrosomorpholine (NNM-stop model). Northern blot analysis revealed increased amounts of G6PDH-mRNA in 9/10 HCC, which are apparently not due to structural alterations of the G6PDH gene as studied by Southern blot analysis. In four additional HCC elevated expression of G6PDH was demonstrated by in situ hybridization to antisense-mRNA and by catalytic enzyme histochemistry. This correlative molecular genetic and enzyme histochemical approach was also used to study G6PDH expression in FAH of different phenotypes, namely glycogen storage foci (GSF), mixed cell foci (MCF) acid basophilic cell foci (BCF), representing early (GSF) and advanced (MCF, BCF) stages of hepatic preneoplasia. mRNA level and activity of G6PDH were closely correlated in all types of lesions and increased from GSF to MCF/BCF and HCC. These results suggest a predominantly transcriptional regulation of the increasing expression of G6PDH during hepatocarcinogenesis in the rat.

Hepatic Preneoplasia in Hepatitis B Virus Transgenic Mice

Hepatocarcinogenesis in hepatitis B virus transgenic mice was studied by means of a correlative cytomorphological and cytochemical approach at different time points in animals from 1 to 34 mo old. HBsAg-positive ground-glass hepatocytes emerged throughout the liver parenchyma in nearly all transgenic mice during the first 4 mo after birth. The panlobular expression of HBsAg persisted until foci of altered hepatocytes appeared (6 to 9 mo of age). Three different types of foci of altered hepatocytes-namely, glycogen-storage foci, mixed cell foci and glycogen-poor foci-developed. Hepatocellular adenomas and carcinomas appeared after 11 mo. Orcein staining revealed frequent transitions between ground-glass hepatocytes extensively expressing HBsAg and glycogen-storage (predominantly clear-cell) foci containing HBsAg-positive cytoplasmic components. Similar transitions between ground-glass hepatocytes and glycogenotic (clear) cells were often found in diffuse parenchymal glycogenosis at 11 or 12 mo. Remnants of HBsAg-positive material were also detected in mixed cell foci, glycogen-poor diffusely basophilic cell foci, hepatic adenoma and hepatocellular carcinoma. These findings suggest that ground-glass hepatocytes are the direct precursor of foci of altered hepatocytes and their neoplastic descendants. The extensive expression of HBsAg is gradually down-regulated during neoplastic transformation, just as the morphological the biochemical phenotypes of foci of altered hepatocytes, hepatic adenoma and hepatocellular carcinoma in transgenic mice resemble those described in chemical hepatocarcinogenesis. The predominant sequence of cellular changes leading from glycogen-storage (predominantly clear cell) foci to mixed cell foci, hepatic adenoma and hepatocellular carcinoma is characterized by a gradual decrease in the activities of glycogen synthase, phosphorylase, glucose-6-phosphatase and adenylate cyclase, whereas glucose-6-phosphate dehydrogenase and pyruvate kinase activities increase. These alterations indicate a shift from the glycogenotic state toward an increase in the pentose phosphate pathway and glycolysis.

Hepatic preneoplasia in hepatitis B virus transgenic mice

Hepatocarcinogenesis in hepatitis B virus transgenic mice was studied by means of a correlative cytomorphological and cytochemical approach at different time points in animals from 1 to 34 mo old. HBsAg-positive ground-glass hepatocytes emerged throughout the liver parenchyma in nearly all transgenic mice during the first 4 mo after birth. The panlobular expression of HBsAg persisted until foci of altered hepatocytes appeared (6 to 9 mo of age). Three different types of foci of altered hepatocytes–namely, glycogen-storage foci, mixed cell foci and glycogen-poor foci–developed. Hepatocellular adenomas and carcinomas appeared after 11 mo. Orcein staining revealed frequent transitions between ground-glass hepatocytes extensively expressing HBsAg and glycogen-storage (predominantly clear-cell) foci containing HBsAg-positive cytoplasmic components. Similar transitions between ground-glass hepatocytes and glycogenotic (clear) cells were often found in diffuse parenchymal glycogenosis at 11 or 12 mo. Remnants of HBsAg-positive material were also detected in mixed cell foci, glycogen-poor diffusely basophilic cell foci, hepatic adenoma and hepatocellular carcinoma. These findings suggest that ground-glass hepatocytes are the direct precursor of foci of altered hepatocytes and their neoplastic descendants. The extensive expression of HBsAg is gradually down-regulated during neoplastic transformation, just as the morphological the biochemical phenotypes of foci of altered hepatocytes, hepatic adenoma and hepatocellular carcinoma in transgenic mice resemble those described in chemical heterocarcinogenesis. The predominant sequence of cellular changes leading from glycogen-storage (predominantly clear cell) foci to mixed cell foci, hepatic adenoma and hepatocellular carcinoma is characterized by a gradual decrease in the activities of glycogen synthase, phosphorylase, glucose-6-phosphatase and adenylate cyclase, whereas glucose-6-phosphate dehydrogenase and pyruvate kinase activities increase. These alterations indicate a shift from the glycogenotic state toward an increase in the pentose phosphate pathway and glycolysis. (Hepatology 1994;20:1162–1172).

Dose and time dependence of the cellular phenotype in rat hepatic preneoplasia and neoplasia induced by single oral exposures to N-nitrosomorpholine.

The dose and time dependence of the cellular phenotype in preneoplastic and neoplastic liver lesions was studied quantitatively in groups of male Sprague-Dawley rats exposed to single oral doses of 0, 200 and 320 mg/kg body wt of N-nitrosomorpholine (NNM) and killed at different time points between 7 and 80 weeks. Compared with the untreated controls, NNM-treated rats showed a dose- and time-dependent increase in the total number and volume of preneoplastic foci of altered hepatocytes (FAH) and in the incidence of hepatocellular adenomas (HCA). In controls, two major phenotypes of FAH were found in low numbers, namely clear and combined clear/acidophilic cell foci which both store excessive amounts of glycogen. The increase in the total number and volume of FAH by single doses of NNM was associated with changes in their cellular phenotype. Clear cell foci were more frequent in the lower dose group than in the higher dose group throughout the observation period. With the exception of the first 15 weeks, combined clear/acidophilic cell foci represented the most frequent phenotype at both dose levels and all time points studied. In contrast, mixed cell foci, which were completely lacking in controls, showed a high relative frequency after a single NNM dose of 320 mg/kg body wt throughout the observation period, but emerged at later time points and in lower numbers when the lower single dose was given. At both dose levels, mixed-cell foci represented the largest phenotype of FAH; their volume fraction correlated positively with the incidence of HCA, indicating a direct precursor-product relationship between these lesions. The size class distribution and temporal appearance of the different phenotypes of FAH and of the adenomas suggest a progression-linked phenotypic instability of the altered cellular phenotypes, resulting in a predominant sequence of cellular changes which leads from glycogenotic clear and acidophilic cell foci to mixed and basophilic cell populations, the latter being poor in glycogen. In addition to these types of FAH, tigroid cell foci, which were very rare in controls, developed in significantly greater numbers after single-dose treatment with NNM at both dose levels. This type of focus may either represent a side lineage of the predominant lineage or an intermediate stage in an alternative lineage of hepatocellular changes, but in any case has the potential to give rise to HCA.

Dose and time dependence of the cellular phenotype in rat hepatic preneoplasia and neoplasia induced by continuous oral exposure to N-nitrosomorpholine.

The dose and time dependence of the cellular phenotype in preneoplastic and neoplastic liver lesions was evaluated quantitatively in groups of male Sprague-Dawley rats continuously exposed to 0, 6, 12 and 24 mg/kg body wt of N-nitrosomorpholine (NNM) and studied at different time points up to 80, 50, 37 and 20 weeks respectively. Continuous oral administration of NNM resulted in a dose- and time-dependent increase in the total number and volume of preneoplastic foci of altered hepatocytes (FAH) and in the incidence of hepatocellular adenomas (HCA) and carcinomas (HCC) at all dose levels studied. In contrast to earlier stop experiments with 24 mg NNM/kg body wt, there was no reversion-linked phenotypic instability but a rapid progression of FAH of the mixed cell type to a high incidence of hepatocyte nodules and HCC after continuous treatment with NNM at this dose level. At the two lower dose levels of NNM (12 and 6 mg/kg) the well-known sequence of cellular changes leading from glycogenotic (clear, combined clear/acidophilic and acidophilic) to mixed and diffusely basophilic cell populations, in which HCC prevailed. A considerable part of the glycogenotic foci contained exclusively acidophilic cells, and HCA consisting of a mixture of acidophilic and basophilic cells were the most common benign tumour type in these groups. At the end of the observation period there was also a high incidence (> 50%) of HCC at both dose levels, indicating the potential of persistent nodules (HCA) containing acidophilic cells to progress to HCC. FAH and HCA exhibiting a tigroid cell pattern appeared only at the two lower dose levels, but for this type of HCA it remained open whether it may progress to HCC. From a comparison of the different dosing regimens of NNM studied in this and previous experiments we conclude that the rapid, potentially reversible shift from glycogenotic to mixed cell populations at the highest dose level of continuously applied NNM (24 mg/kg) and the high proportion of pure acidophilic glycogen storage foci observed after continuous administration of NNM at the two lower dose levels (6 and 12 mg/kg) represent different phenotypic expressions of promoting effects exerted by the ongoing influence of the carcinogen on FAH initiated by the same compound. The metabolic and molecular changes underlying these 'initiating' and 'promoting' effects of NNM seem to differ in terms of quantity rather than quality.

Dose and time dependence of the cellular phenotype in rat hepatic preneoplasia and neoplasia induced in stop experiments by oral exposure to N-nitrosomorpholine.

The dose and time dependence of the cellular phenotype in preneoplastic and neoplastic liver lesions was evaluated quantitatively in groups of male Sprague-Dawley rats exposed for 7 weeks to 0, 12 and 24 mg/kg body wt of N-nitrosomorpholine (NNM) and studied at different time points up to 80 weeks after withdrawal of NNM (stop model). NNM-treated rats showed a dose- and time-dependent increase in the total number and volume of preneoplastic foci of altered hepatocytes (FAH) and in the incidence of hepatocellular adenomas (HCA) and carcinomas (HCC) at both dose levels, compared with the untreated controls. After stopping treatment with 12 mg/kg body wt, the well-known sequence of cellular changes leading from glycogenotic clear and clear/acidophilic cell foci to mixed and diffusely basophilic cell populations poor in glycogen was found. In contrast, at the higher NNM dose level (24 mg/kg) predominantly mixed and diffusely basophilic cell foci appeared immediately after cessation of treatment, but their number rapidly declined up to 13 weeks after withdrawal. At the same time, there was a reciprocal increase in the number of the less altered clear/acidophilic cell foci, indicating an early reversion-linked phenotypic instability of FAH. However, in spite of this reversion higher numbers of mixed and diffusely basophilic cell foci were retained after treatment with 24 compared to 12 mg/kg of NNM at all time points studied, and there was even a slow additional increase in the number of these types of FAH 20 weeks after withdrawal of NNM. At both dose levels, the volume fraction of the persistent mixed cell foci correlated positively with the incidence of HCA and HCC, suggesting that this phenotype of FAH represents a direct precursor of the neoplastic lesions. Tigroid cell foci, which appeared most frequently after treatment with the lower dose of NNM, were not integrated into the predominant sequence of cellular changes leading to HCC, but they may represent an intermediate stage in a side lineage of this sequence, endowed with the potential to progress at least to HCA. Our results show that reversion-linked phenotypic instability of FAH occurs mainly after high dose treatment, possibly resulting from rapid adaptive cellular responses to the primary carcinogenic lesion(s) which may be fixed by genetic or epigenetic mechanisms. In contrast, progression-linked phenotypic instability of FAH is a slow process developing in a dose- and time-dependent manner at all dose levels leading to hepatic neoplasia.

Two-Year Toxicity Study of Doxylamine Succinate in B6C3F1 Mice

Doxylamine succinate, a commonly used antihistamine, was administered as an admixture in the feed to groups of male and female B6C3F1 mice at dosage levels of 0,190,375, and 750 parts per million (ppm) (based on free amine) for 65 weeks (12 per group) or 2 years (48 per group). Survival to terminal sacrifice in the 2-year groups was 85–98% with no significant differences between groups of the same sex. Final body weights of the highest dose group were 3.4% and 8.7% less than controls in males and females, respectively. Doxylamine produced liver lesions in male mice including hepatocellular hypertrophy, atypical hepatocytes, clear cell and mixed cell foci, and necrosis. In females, doxylamine produced liver fatty change, hepatocellular hypertrophy, and necrosis. Doxylamine produced a significant increase in hepatocellular adenomas in the mid- and high-dosage groups of males and in the high-dosage group of females. Thyroid follicular cell hyperplasia and thyroid follicular cell adenomas also were increased in treated mice of both sexes. A treatment-related increase in cytoplasmic alteration of the parotid salivary gland in males and an increased incidence in hyperplasia of the pituitary gland in females were observed.