Foci Of Altered Hepatocytes Research Articles

METTL3 Regulates Liver Homeostasis, Hepatocyte Ploidy, and Circadian Rhythm–Controlled Gene Expression in Mice

N6-methyladenosine (m6A), the most abundant internal modifier of mRNAs installed by the methyltransferase 13 (METTL3) at the (G/A)(m6A)C motif, plays a critical role in the regulation of gene expression. METTL3 is essential for embryonic development, and its dysregulation is linked to various diseases. However, the role of METTL3 in liver biology is largely unknown. In this study, METTL3 function was unraveled in mice depleted of Mettl3 in neonatal livers (Mettl3fl/fl; Alb-Cre). Liver-specific Mettl3 knockout (M3LKO) mice exhibited global decrease in m6A on polyadenylated RNAs and pathologic features associated with nonalcoholic fatty liver disease (eg, hepatocyte ballooning, ductular reaction, microsteatosis, pleomorphic nuclei, DNA damage, foci of altered hepatocytes, focal lobular and portal inflammation, and elevated serum alanine transaminase/alkaline phosphatase levels). Mettl3-depleted hepatocytes were highly proliferative, with decreased numbers of binucleate hepatocytes and increased nuclear polyploidy. M3LKO livers were characterized by reduced m6A and expression of several key metabolic transcripts regulated by circadian rhythm and decreased nuclear protein levels of the core clock transcription factors BMAL1 and CLOCK. A significant decrease in total Bmal1 and Clock mRNAs but an increase in their nuclear levels were observed in M3LKO livers, suggesting impaired nuclear export. Consistent with the phenotype, methylated (m6A) RNA immunoprecipitation coupled with sequencing and RNA sequencing revealed transcriptome-wide loss of m6A markers and alterations in abundance of mRNAs involved in metabolism in M3LKO. Collectively, METTL3 and m6A modifications are critical regulators of liver homeostasis and function.

Combining In Vivo and Organotypic In Vitro Approaches to Assess the Human Relevance of Basimglurant (RG7090), a Potential CAR Activator.

Basimglurant (RG7090), a small molecule under development to treat certain forms of depression, demonstrated foci of altered hepatocytes in a long-term rodent-toxicity study. Additional evidence pointed toward the activation of the constitutive androstane receptor (CAR), an established promoter of nongenotoxic and rodent-specific hepatic tumors. This mode of action and the potential human relevance was explored in vivo using rodent and cynomolgus monkey models and in vitro using murine and human liver spheroids. Wild type (WT) and CAR/pregnane X receptor (PXR) knockout mice (CAR/PXR KO) were exposed to RG7090 for 8 consecutive days. Analysis of liver lysates revealed induction of Cyp2b mRNA and enzyme activity, a known activation marker of CAR, in WT but not in CAR/PXR KO animals. A series of proliferative genes were upregulated in WT mice only, and immunohistochemistry data showed increased cell proliferation exclusively in WT mice. In addition, primary mouse liver spheroids were challenged with RG7090 in the presence or absence of modified antisense oligonucleotides inhibiting CAR and/or PXR mRNA, showing a concentration-dependent Cyp2b mRNA induction only if CAR was not repressed. On the contrary, neither human liver spheroids nor cynomolgus monkeys exposed to RG7090 triggered CYP2B mRNA upregulation. Our data suggested RG7090 to be a rodent-specific CAR activator, and that CAR activation and its downstream processes were involved in the foci of altered hepatocytes formation detected in vivo. Furthermore, we demonstrated the potential of a new in vitro approach using liver spheroids and antisense oligonucleotides for CAR knockdown experiments, which could eventually replace in vivo investigations using CAR/PXR KO mice.

First approaches for the transplantation of hepatocytes from Wistar rat preneoplastic livers into healthy recipients.

the shortage of donors of hepatocyte transplantation therapy led to the use of so-called marginal donors. Some donors may have a hepatic illnesses that is associated with hepatic preneoplasia with foci of altered hepatocytes (FAH). to determine whether recipients developed FAH upon transplantation with hepatocytes from a preneoplastic liver and whether FAH progresses to a preneoplastic hepatocyte-derived tumor (PHDT), up to 60 days after transplantation. male Wistar adult rats were used as donors and recipients. Donors underwent a 2-phase model of liver preneoplasia for hepatocyte isolation. Recipients underwent a partial two thirds hepatectomy and received 150,000 hepatocytes. Recipients were euthanized seven and 60 days after transplantation. The number of FAH per liver area, percentage of liver occupied by FAH, the hepatic enzymatic profile, the percentage of prothrombin time (PT), the proliferative index (PI) and liver morphology were analyzed. recipients developed few and very isolated FAH. No statistical differences were found between hepatic enzyme activities and PT. There were no differences between the groups with regard to the number of FAH per liver area and percentage of liver occupied by FAH after 60 days. The PI decreased on day 60 compared to day seven. No morphological alterations were found. recipients developed few FAH that did not increase in number or size, nor did they progress to PHDT and had normal plasma biochemical features and liver morphology up to 60 days post-transplant. Additional studies are needed to determine whether FAH development constitutes a risk for recipients while waiting for whole organ transplant.

In ovo hepatocarcinogenicity of N-nitrosodimethylamine and N-nitrosodimethylamine in White Leghorn chickens

Avian embryos have been gaining an increasing scientific interest as a valuable model system for the experimental cancer research that could contribute to a significant reduction of the number of laboratory animals. In the present study, the liver lesions induced by N-nitrosodimethylamine and N-nitrosodiethylamine in 15I line, White Leghorn embryos were identified and studied by routine histopathological method s. Foci of altered hepatocytes with basophilic and eosinophilic phenotype, well known as preneoplastic alterations were identified in the avian embryonal livers after in ovo exposure to both N-nitroso compounds. These studies were further extended by histopathological, haematological and biochemical examinations on the effects of N-nitrosodimethylamine in chickens hatched from carcinogen-inoculated eggs. In addition to the preneoplastic lesions observed in the avian livers, proliferations of oval and hepatocellular carcinoma cells, with clearly expressed signs of malignancy were found. The in ovo application of the chemical carcinogen was found to affect both hematological and blood biochemistry parameters measured in experimental birds. The established conditions such as thrombocytopenia and increased levels of liver enzymes, as an essential part of the paraneoplastic syndrome, were associated with the process of hepatocarcinogenesis. The results of this study confirm the preneoplastic nature of the focal lesions in embryonal avian liver and their progression to liver neoplastic alterations after a single in ovo application of known hepatocarcinogens. Moreover, the results indicate that 15I line, White Leghorn embryos are anew, valuable in ovo model for studies on hepatocarcinogenicity of chemical compounds and underline the importance of research on the development of different avian models of carcinogenicity.

Fibrosis-associated hepatocarcinogenesis revisited: Establishing standard medium-term chemically-induced male and female models.

Hepatocellular carcinoma causes ~10% of all cancer-related deaths worldwide, usually emerging in a background of liver fibrosis/cirrhosis (70%-90% of cases). Chemically-induced mouse models for fibrosis-associated hepatocarcinogenesis are widely-applied, resembling the corresponding human disease. Nonetheless, a long time is necessary for the development of preneoplastic/neoplastic lesions. Thus, we proposed an early fibrosis-associated hepatocarcinogenesis model for male and female mice separately, focusing on reducing the experimental time for preneoplastic/neoplastic lesions development and establishing standard models for both sexes. Then, two-week old susceptible C3H/HeJ male and female mice (n = 8 animals/sex/group) received a single dose of diethylnitrosamine (DEN, 10 or 50 mg/Kg). During 2 months, mice received 3 weekly doses of carbon tetrachloride (CCl4, 10% corn oil solution, 0.25 to 1.50 μL/g b.wt.) and they were euthanized at week 17. DEN/CCl4 protocols for males and females displayed clear liver fibrosis, featuring collagen accumulation and hepatic stellate cell activation (α-SMA). In addition, liver from males displayed increased CD68+ macrophage number, COX-2 protein expression and IL-6 levels. The DEN/CCl4 models in both sexes impaired antioxidant defense as well as enhanced hepatocyte proliferation and apoptosis. Moreover, DEN/CCl4-treated male and female developed multiple preneoplastic altered hepatocyte foci and hepatocellular adenomas. As expected, the models showed clear male bias. Therefore, we established standard and suitable fibrosis-associated hepatocarcinogenesis models for male and female mice, shortening the experimental time for the development of hepatocellular preneoplastic/neoplastic lesions in comparison to other classical models.

Evaluation of the effect of Gallic acid against carbon tetrachloride-induced liver injury in albino rats: histological, immunohistochemical and biochemical study

Introduction: Carbon tetrachloride (CCL4) is used for induction of liver fibrosis. Liver fibrosis has no standard treatment. Gallic acid (GA) is a naturally occurring phenolic compound having anti-inflammatory and anti-cancer activities.Aim of the work: This work aimed to evaluate the preventive role of GA against CCL4 induced liver fibrosis.Materials and Methods: Forty adult male albino rats were divided into 4 groups; Group I: without treatment .GroupII: received olive oil. Group III: received 1.5 mL/kg of CCL4 twice a week. Group IV: received CCL4 with GA 100mg/kg. Animals were sacrificed after 8 weeks. Liver specimens were processed for light and electron microscopic (EM) study. Area % of collagen and α-SMA expression, levels of AST, ALT, ALP, reduced GSH, MDA, SOD and hydroxyproline were measured and evaluated statistically.Results: Group III revealed foci of altered hepatocytes with dense nuclei and vacuolated cytoplasm. Dilatation and congestion of central and portal veins with mononuclear cell infiltration were observed. There was increased collagen deposition around the central vein. Intense stained α-SMA-positive cells were observed. CCL4 increased collagen and α-SMA expression area %. EM showed electronlucent areas in the cytoplasm of the hepatocytes, vacuolation and margination of nuclear chromatin. CCL4 increased AST, ALT, ALP, hepatic MDA and hydroxyproline levels. Moreover, it decreased the activities of SOD and reduced GSH. The co-administration of GA prevented most of these histological and biochemical changes.Conclusions: The use of natural antioxidants as GA can be promising in ameliorating liver fibrosis better than the drugs and their side effects.

Use of early phenotypic in vivo markers to assess human relevance of an unusual rodent non-genotoxic carcinogen in vitro.

Foci of altered hepatocytes (FAH) are considered putative, pre-neoplastic lesions that can occur spontaneously in aging rodents, but can also be induced by chemicals or drugs. Progression of FAH to hepatocellular neoplasms has been reported repeatedly but increases in foci in rodents do not necessarily lead to tumors in carcinogenicity studies and the relevance for humans often remains unclear. Here we present the case of RG3487, a molecule which induced FAH and, later on, tumors in rats. Because the molecule was negative in genotoxicity assays it was classified as a non-genotoxic carcinogen. In order to assess the potential for liver tumor formation in humans, we analyzed treatment-induced changes in vivo to establish a possible mode of action (MoA). In vivo and in vitro gene expression analysis revealed that nuclear receptor signaling was unlikely to be the relevant MoA and no other known mechanism could be established. We therefore took an approach comparing phenotypic markers, including mRNA changes, proliferation and glycogen accumulation, in vitro using cells of different species to assess the human relevance of this finding. Since the alterations observed in rats were not seen in the liver of mice or dogs in vivo, we could validate the relevance of the cell models chosen by use of hepatocytes from these species in vitro. This ultimately allowed for a cross-species comparison, which suggested that the formation of FAH and liver tumors was rat specific and unlikely to translate to human. Our work showed that phenotypic species comparison in vitro is a useful approach for assessment of the human relevance of pre-clinical findings where no known mechanism can be established.

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.

Ptgr1 expression is regulated by NRF2 in rat hepatocarcinogenesis and promotes cell proliferation and resistance to oxidative stress

Prostaglandin reductase-1 (Ptgr1) is an alkenal/one oxidoreductase that is involved in the catabolism of eicosanoids and lipid peroxidation such as 4-hydroxynonenal (4-HNE). Recently, we reported that Ptgr1 is overexpressed in human clinical and experimentally induced samples of hepatocellular carcinoma (HCC). However, how the expression of this gene is regulated and its role in carcinogenesis are not yet known. Here, we studied parameters associated with antioxidant responses and the mechanisms underlying the induction of Ptgr1 expression by the activation of Nuclear Factor (erythroid-derived-2)-like-2 (NRF2). For these experiments, we used two protocols of induced hepatocarcinogenesis in rats. Furthermore, we determined the effect of PTGR1 on cell proliferation and resistance to oxidative stress in cell cultures of the epithelial liver cell line, C9. Ptgr1 was overexpressed during the early phase in altered hepatocyte foci, and this high level of expression was maintained in persistent nodules until tumors developed. Ptgr1 expression was regulated by NRF2, which bound to an antioxidant response element at −653bp in the rat Ptgr1 gene. The activation of NRF2 induced the activation of an antioxidant response that included effects on proteins such as glutamate-cysteine ligase, catalytic subunit, NAD(P)H dehydrogenase quinone-1 (NQO1) and glutathione-S-transferase-P (GSTP1). These effects may have produced a reduced status that was associated with a high proliferation rate in experimental tumors. Indeed, when Ptgr1 was stably expressed, we observed a reduction in the time required for proliferation and a protective effect against hydrogen peroxide- and 4-HNE-induced cell death. These data were consistent with data showing colocalization between PTGR1 and 4-HNE protein adducts in liver nodules. These findings suggest that Ptgr1 and antioxidant responses act as a metabolic adaptation and could contribute to proliferation and cell-death evasion in liver tumor cells. Furthermore, these data indicate that Ptgr1 could be used to design early diagnostic tools or targeted therapies for HCC.

The Epidermal Growth Factor Receptor (EGFR) Inhibitor Gefitinib Reduces but Does Not Prevent Tumorigenesis in Chemical and Hormonal Induced Hepatocarcinogenesis Rat Models.

Activation of the epidermal growth factor receptor (EGFR) signaling pathway promotes the development of hepatocellular adenoma (HCA) and carcinoma (HCC). The selective EGFR inhibitor Gefitinib was found to prevent hepatocarcinogenesis in rat cirrhotic livers. Thus, Gefitinib might reduce progression of pre-neoplastic liver lesions to HCC. In short- and long-term experiments, administration of N-Nitrosomorpholine (NNM) or intrahepatic transplantation of pancreatic islets in diabetic (PTx), thyroid follicles in thyroidectomized (TTx) and ovarian fragments in ovariectomized (OTx) rats was conducted for the induction of foci of altered hepatocytes (FAH). Gefitinib was administered for two weeks (20 mg/kg) or three and nine months (10 mg/kg). In NNM-treated rats, Gefitinib administration decreased the amount of FAH when compared to controls. The amount of HCA and HCC was decreased, but development was not prevented. Upon all transplantation models, proliferative activity of FAH was lower after administration of Gefitinib in short-term experiments. Nevertheless, the burden of HCA and HCC was not changed in later stages. Thus, EGFR inhibition by Gefitinib diminishes chemical and hormonal also induced hepatocarcinogenesis in the initiation stage in the non-cirrhotic liver. However, progression to malignant hepatocellular tumors was not prevented, indicating only a limited relevance of the EGFR signaling cascade in later stages of hepatocarcinogenesis.

Sequential dietary exposure to aflatoxin B1 and fumonisin B1 in F344 rats increases liver preneoplastic changes indicative of a synergistic interaction

Dietary co-exposure to aflatoxin B1 (AFB1) and fumonisin B1 (FB1) and their interaction on hepatocellular carcinogenesis is of particular concern in toxicology and public health. In this study we evaluated the liver preneoplastic effects of single and sequential dietary exposure to AFB1 and FB1 in the F344 rat carcinogenesis model. Serum biochemical alterations, liver histopathological changes, and the formation of liver glutathione S transferase positive (GST-P+) foci were the major outcome parameters examined. Compared to the AFB1-only treatment, the FB1-only treatment induced less dysplasia, and more apoptosis and mitoses. Sequential AFB1 and FB1 treatment lead to increased numbers of dysplasia, apoptosis and foci of altered hepatocytes, as compared to either mycotoxin treatment alone. More importantly, sequential exposure to AFB1 and FB1 synergistically increased the numbers of liver GTP-P+ foci by approximately 7.3-and 12.9-fold and increased the mean sizes of GST-P+ foci by 6- and 7.5-fold, respectively, as compared to AFB1- or FB1-only treatment groups. In addition, liver ALT and AST levels were significantly increased after sequential treatment as compared to single treatment groups. The results demonstrate the interactive effect of dietary AFB1 and FB1 in inducing liver GST-P+ foci formation and provide information to model future intervention studies.

Utilizing Whole Slide Images for Pathology Peer Review and Working Groups.

This article describes the results of comparisons of digitally scanned whole slide images (WSIs) and glass microscope slides for diagnosis of tissues under peer review by the National Toxicology Program. Findings in this article were developed as a result of the data collected from 6 pathology working groups (PWGs), 1 pathology peer review, and survey comments from over 25 participating pathologists. For each PWG, 6-14 pathologists examined 10-143 tissues per study from 6- and 9-month perinatal studies and 2-year carcinogenicity studies. Overall it was found that evaluation of WSIs is generally equivalent to using glass slides. Concordance of PWG consensus diagnoses based upon review of WSIs versus glass slides ranged from 74% to 100% (median 86%). The intra- and interobserver diagnostic variation did not appear to influence the conclusions of any study. Based upon user opinions collected from surveys, WSIs may be less optimal than glass slides for evaluation of subtle lesions, large complex lesions, small lesions in a large section of tissue, and foci of altered hepatocytes. These results indicate that, although there may be some limitations, the use of WSIs can effectively accomplish the objectives of a conventional glass slide review and definitely serves as a useful adjunct to the conduct of PWGs.

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.

Activation of oxidative stress and inflammatory factors could account for histopathological progression of aflatoxin-B1 induced hepatocarcinogenesis in rat.

Aflatoxin-B1 (AFB1) intoxication is known to develop hepatocellular carcinoma (HCC). However, pathogenesis and diagnosis of AFB1-induced HCC remain undefined. This article describes histopathological progression versus kinetics of the placental glutathione S-transferase (GST-pi) expression and profiles of the antioxidant enzymes, pro-inflammatory cytokines, and proliferative cell nuclear antigen (PCNA) in the liver from the rats administered with two doses of 1 mg AFB1/kg b.w. Histopathologically, hepatocytes necrosis could be observed after 4 weeks of the AFB1 treatment, which subsequently developed into the well-defined foci of altered hepatocytes (FAH) at 10 weeks post-treatment stage. This was consistent with an increasing trend of GST-pi immunostaining especially in the liver foci as a function of FAH progression and thus, suggesting that GST-pi over expression may serve as a marker for AFB1-induced hepatocarcinogenesis. The liver from AFB1-treated rats showed significantly enhanced level of reactive oxygen species coinciding with the declined immunostaining for superoxide dismutase-1, a committed enzyme of the antioxidant pathway, in the FAH regions and also with declined activity of the other antioxidant enzymes. Concordantly, the liver from the AFB1-treated rats showed over expression of pro-inflammatory cytokines; TNF-α & IL-1α and a cell proliferative marker PCNA. These findings present histological characterization of AFB1-induced HCC development and provide evidence for activation of oxidative stress-pro-inflammatory pathway during hepatocarcinogenesis induced by AFB1 toxicity.

Abstract 4946: Developing anti-angiogenic therapies for human hepatocellular cancer (HCC)- studies of suntinib in the woodchuck model of hepatitis B related HCC

Abstract Introduction: Better animal models that recapitulate the liver milieu of human HCC are needed. The eastern woodchuck is an established model of human hepatitis B viral infection and spontaneously develops HCC in the context of chronic woodchuck hepatitis viral infection (WHV). The translational relevance of this model for developing anti-angiogenic therapies was evaluated using sunitinib (S), a potent oral, anti-angiogenic agent. Methods: Woodchucks were bred and inoculated at birth with titered infectious WHV pools obtained from chronic WHV carriers. By 12 months of age, the rate of chronic WHV infection was >60%. Carriers were followed by USG, upon developing HCC, 12 animals were randomized 1:1 to S or placebo (P) given once orally daily for 30 days. From a single treatment S PK study at 4 dose levels, n=3/group, simulations showed 12mg/kg daily was expected to be optimal for achieving steady state serum concentrations between 50- 100 ng/ml in woodchucks. Tumor size and blood flow were assessed using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) before treatment and on day 28 using standardized protocols. At study completion or when animals were humanely euthanized, tumors and any other small nodules were fixed overnight in 10% buffered formalin. After standard processing and embedding in paraffin, 4 μm sections were prepared, deparaffinized, stained with hematoxylin-eosin (H&E), studied with a Zeiss Axio Imager A1 microscope by a pathologist blinded to the treatment arm. Morphometric study of areas of necrosis were compared to the size of the tumor sections by counting view fields at medium objective magnification, and differences in percentages between the groups were compared by the Excel student's two tailed, two-sample unequal variance t-test. Results: The median age was 30 months. Four animals died during the course of the study (1P, 3 S) and were replaced. Median therapy duration was 28 days. Morphologically, portal hepatitis and pre-neoplastic lesions (foci of altered hepatocytes [FAH]) and larger areas of altered hepatocytes (AAH) were seen in all livers. Areas of tumor necrosis of varying size (20-100%) adjacent to terminal hepatic venules seen in both high grade and in rare, low grade tumor sections of S treated woodchucks were rare in P animals. The difference was highly significant (p= 6.0 E-13) between the two groups. By DCE-MRI, median tumor volume change was + 17% and -13% and necrotic tumor volume change was +22% and +190% in P and S treated animals respectively. However all MRI parameters (k trans, AUC90, median tumor volume) were not statistically significantly different between the groups. Conclusions: Angiogenic response across different stages of hepatocarcinogenesis can be studied in woodchucks. Histological necrosis in S treated woodchucks without significant DCE-MRI change and the toxicities seen with S have translational significance to human HCC. Citation Format: Alexander Pomakov, Ilia Toshkov, Sandra Buitrago, Leslie Curtin, Donald Trump, Candace Johnson, Edward Ashton, Bud Tennant, Renuka Iyer. Developing anti-angiogenic therapies for human hepatocellular cancer (HCC)- studies of suntinib in the woodchuck model of hepatitis B related HCC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4946. doi:10.1158/1538-7445.AM2014-4946

Liver pro-oncogenic potential of SERPINB3.

Hepatocellular Carcinoma (HCC) is a growing sanitary problem worldwide. This tumor is indeed the third most deadly and the fifth most common cancer all over the word, with an incidence that, at least in the United States, has doubled in the past two decades [1]. Current studies suggest that HCC can derive from liver progenitor cells or cancer stem cells (CSCs) in which signaling pathway similar to those identified in liver cancer (e.g., Wnt, TGF-β, Notch, Hedgehog, and PI3K/AKT/mTOR), are activated. These findings support the hypothesis that CSCs contribute to the molecular heterogeneity and chemoresistance of this tumor [2]. In addition, the progenitor-like subtype of hepatocellular carcinoma is associated with a poor prognosis [2]. Cancer stem cells are neoplastic cells that possess distinct survival mechanisms and stem cell properties responsible for tumor formation and progression. Although the origin of CSCs in liver cancer is not fully elucidated, has been recently demonstrated that any hepatic lineage cell (tumor progenitor cells, stem cells, or dedifferentiated cells) can be reprogrammed into CSC by activating diverse cell type-specific pathways [3]. The role of preneoplastic lesions in HCC remains still controversial. A recent study [4] has demonstrated that in foci of altered hepatocytes (FAH), consisting in highly proliferative cells, compared to the parenchyma of cirrhotic liver, resides aggregates of normal hepatocytes, oval cells and progenitor cells with high expression of specific markers, including CD90, CD44, EpCAM and alpha-fetoprotein (AFP). It was further demonstrated that this subset of cells with stem/progenitor are tumorigenic in damaged livers (i.e., in chronic injury and compensatory proliferation) and in this context the microenvironment, IL-6 and TGF-β signaling are required to promote in vivo growth and malignant progression of hepatic cancer progenitor cells and tumor metastasis [4]. In the liver, the ov-serpin SERPINB3 (or SCCA1) and its isoforms SERPINB4 (or SCCA2) are undetectable in normal hepatocytes, but their expression progressively increases in chronic liver diseases, dysplastic nodules and HCC, suggesting that they may be involved in relatively early events of hepatocarcinogenesis, although their specific role has not been defined yet [5]. In the article “SERPINB3 is associated with TGF-β1 and cytoplasmic β-catenin expression in hepatocellular carcinomas with poor prognosis” [5] Turato et al, for the first time, provided evidence that in liver tumors the expression of the protease inhibitor SERPINB3 was correlated with the molecular markers of poor prognosis, (TGF-β signalling associated with Wnt target gene expression). High expression of SERPINB3 was indeed significantly associated with early tumor recurrence, which is commonly regarded as a true metastasis, due to dissemination of primary tumor cells then representing a subset of most aggressive HCCs. These results are in agreement with previous studies in vitro, demonstrating that SERPINB3 can enhance TGF-β expression and induce epithelial-mesenchymal transition, with increased invasiveness and cell proliferation [5]. Figure 1 Proposed involvement of SERPINB3 in liver carcinogenesis The presence of SERPINB3 in human progenitor cells compartment has been recently demonstrated by our group. This serpin was indeed detected in EpCAM + cell fractions sorted from human foetal and adult livers and in the same ductular structures that express stem/progenitor cell markers. Moreover, the mouse homologous, Serpinb3b, was induced in a mouse model of liver stem/progenitor cell activation [6]. Expression of SERPINB3 protein was also observed in hepatoblastoma, considered an embryonal tumour of the liver, especially in tumors with more advanced stage. The intensity of the protein signal was higher in less differentiated components of the tumor and in the clusters of undifferentiated variant, named small undifferentiated cell (SCUD) pattern, considered as primitive uncommitted progenitor cells [7]. Recent findings revealed that SERPINB3/4 isoforms are RAS-responsive factors that can lead to inflammatory protein production and tumorigenesis. They are transcriptionally upregulated via MAPK and the ETS family transcription factors PEA3. The increased expression of these serpin family members determines NF-kB activation, essential for RAS-mediated cytokine production and tumor growth. The positive correlation between Ras mutation, enhanced SERPINB3 expression and IL-6 expression was documented in human colorectal and pancreatic tumour samples, reflecting the inflammatory and protumorigenic role of this serpin [8]. These last results, taken together with the finding that high SERPINB3 expression was detected in liver progenitor cells and in the most aggressive liver tumors [5,7] suggest that this serpin, may determine a pro-tumorogenic microenvironment milieu and cellular features with a high pro-oncogenic potential.

Dose-dependent induction of preneoplastic lesions by the tobacco-specific nitrosamine carcinogen NNK in the in ovo carcinogenicity assessment (IOCA) assay

The potential of the carcinogenic tobacco specific nitrosamine 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-1-butanone (NNK) to induce preneoplastic hepatocellular altered foci (HAF) was tested in the in ovo carcinogenicity assessment (IOCA) assay. Single doses of NNK over a dose range from 0.1mg to 6mg were injected into fertilized turkey eggs prior to incubation for 24 days. The livers were investigated by histological, histochemical and morphometric methods. Mortality was increased for eggs exposed to 6mg. In this group, the whole livers were severely altered, showing pronounced changes of nucleus size and signs of cell death. At the dose of 2mg various types of foci of altered hepatocytes (HAF) were observed. Basophilic cell foci of the solid or tubular type were most frequent. The NNK-induced HAF were very similar to the preneoplastic lesions that occur in the livers of mammals during hepatocarcinogenesis which are regarded as early indicators of carcinogenesis. The similarity to the HAF in rodents included histochemically detectable alterations like decreased activities of glucose-6-phosphatase, adenosine triphosphatase and glycogen phosphorylase. At doses of 1mg or below, no HAF were detected. At all dose levels an increased occurrence of enlarged hepatocytes with enlarged nuclei and prominent nucleoli (karyomegalic hepatocytes) were observed. The increase in karyomegalic hepatocytes was also statistically significant at the low dose of 0.1mg/kg NNK but the dose–effect curve for their induction was clearly non-linear. Induction of HAF and karyomegalic hepatocytes in ovo is a simple (one dose), rapid (24 days) and inexpensive (no animal purchase or housing) experimental approach for studies on chemically induced hepatocarcinogenesis.

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.

Clear cell foci of altered hepatocytes and hepatocellular carcinomas in the N-Nitrosomorpholine (NNM) model in the rat show an overexpression of pro-oncogenic pathways and metabolic alterations similar to lesions in the islet-transplantation model

Background and aims: Administration of the N-Nitrosamine N-Nitrosomorpholine (NNM) in rats is a well examined model of chemically induced hepatocarcinogenesis and starts with the formation of focal pre-neoplastic lesions in the liver, i.e. clear cell foci (CCF) of altered hepatocytes, and induces the formation of hepatocellular adenomas (HCA) and carcinomas (HCC). Similar lesions are known in the rat model of insulin-induced hepatocarcinogenesis and also in the human liver, which reveal an activation of the AKT/mTOR and Ras/MAPK pathways and the lipogenic phenotype, as we described previously.

Proceedings of the 2012 National Toxicology Program Satellite Symposium.

The 2012 annual National Toxicology Program (NTP) Satellite Symposium, entitled "Pathology Potpourri," was held in Boston in advance of the Society of Toxicologic Pathology's 31st annual meeting. The goal of the NTP Symposium is to present current diagnostic pathology or nomenclature issues to the toxicologic pathology community. This article presents summaries of the speakers' presentations, including diagnostic or nomenclature issues that were presented, along with select images that were used for audience voting or discussion. Some lesions and topics covered during the symposium include eosinophilic crystalline pneumonia in a transgenic mouse model; differentiating adrenal cortical cystic degeneration from adenoma; atypical eosinophilic foci of altered hepatocytes; differentiating cardiac schwannoma from cardiomyopathy; diagnosis of cardiac papillary muscle lesions; intrahepatocytic erythrocytes and venous subendothelial hepatocytes; lesions in Rathke's cleft and pars distalis; pernicious anemia and megaloblastic disorders; embryonic neuroepithelial dysplasia, holoprosencephaly and exencephaly; and INHAND nomenclature for select cardiovascular lesions.