Chronically high level of tgfb1a induction causes both hepatocellular carcinoma and cholangiocarcinoma via a dominant Erk pathway in zebrafish.

Liver cancers including both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) have increased steadily with the prevalence of non-alcoholic steatohepatitis (NASH), but the underlying mechanism for the transition from NASH to liver cancers remains unclear. Here we first employed diet-induced NASH zebrafish and found that elevated level of satiety hormone, leptin, induced overexpression of tgfb1. Then we developed tgfb1a transgenic zebrafish for inducible, hepatocyte-specific expression. Interestingly, chronically high tgfb1a induction in hepatocytes could concurrently drive both HCC and CCA. Molecularly, oncogenicity of Tgfb1 in HCC was dependent on the switch of dominant activated signaling pathway from Smad to Erk in hepatocytes while concurrent activation of both Smad and Erk pathways in cholangiocytes was essential for Tgfb1-induced CCA. These findings pinpointed the novel role of Tgfb1 as a central regulator in the two major types of liver cancers, which was also supported by human liver disease samples.

Stress Leukogram Induced by Acute and Chronic Stress in Zebrafish (Danio rerio).

The use of zebrafish (Danio rerio) as an animal model for experimental studies of stress has increased rapidly over the years. Although many physiologic and behavioral characteristics associated with stress have been defined in zebrafish, the effects of stress on hematologic parameters have not been described. The purpose of our study was to induce a rise in endogenous cortisol through various acute and chronic stressors and compare the effects of these stressors on peripheral WBC populations. Acutely stressed fish underwent dorsal or full-body exposure to air for 3 min, repeated every 30 min over the course of 90 min. Chronically stressed fish underwent exposure to stressors twice daily over a period of 5 d. After the last stressful event, fish were euthanized, and whole blood and plasma were obtained. A drop of whole blood was used to create a blood smear, which was subsequently stained with a modified Wright-Giemsa stain and a 50-WBC differential count determined. Plasma cortisol levels were determined by using a commercially available ELISA. Endogenous cortisol concentrations were significantly higher in both stressed groups as compared with control fish. Acutely stressed fish demonstrated significant lymphopenia, monocytosis, and neutrophilia, compared with unstressed, control fish. Chronic stress induced lymphopenia and monocytosis but no significant changes in relative neutrophil populations in zebrafish. The changes in both stressed groups most likely are due to increases in endogenous cortisol concentrations and represent the first description of a stress leukogram in zebrafish.

Males develop faster and more severe hepatocellular carcinoma than females in krasV12 transgenic zebrafish.

Hepatocellular carcinoma (HCC) is more prevalent in men than women, but the reason for this gender disparity is not well understood. To investigate whether zebrafish could be used to study the gender disparity of HCC, we compared the difference of liver tumorigenesis between female and male fish during early tumorigenesis and long-term tumor progression in our previously established inducible and reversible HCC model - the krasV12 transgenic zebrafish. We found that male fish developed HCC faster than females. The male tumors were more severe from the initiation stage, characteristic of higher proliferation, activation of WNT/ß-catenin pathway and loss of cell adhesion. During long-term tumor progression, the male tumors developed into more advanced multi-nodular tumors, whereas the female tumors remain uniform and homogenous. Moreover, regression of male tumors required longer time. We further investigated the role of sex hormones in krasV12 transgenic fish. Estrogen treatment showed tumor suppressing effect during early tumorigenesis through inhibiting cell proliferation, whereas androgen accelerated tumor growth by promoting cell proliferation. Overall, our study presented the zebrafish as a useful animal model for study of gender disparity of HCC.

Development of a conditional liver tumor model by mifepristone-inducible Cre recombination to control oncogenic kras V12 expression in transgenic zebrafish.

Here we report a new transgenic expression system by combination of liver-specific expression, mifepristone induction and Cre-loxP recombination to conditionally control the expression of oncogenic kras(V12). This transgenic system allowed expression of kras(V12) specifically in the liver by a brief exposure of mifepristone to induce permanent genomic recombination mediated by the Cre-loxP system. We found that liver tumors were generally induced from multiple foci due to incomplete Cre-loxP recombination, thus mimicking naturally occurring human tumors resulting from one or a few mutated cells and clonal proliferation to form nodules. Similar to our earlier studies by both constitutive and inducible expression of the kras(V12) oncogene, hepatocellular carcinoma (HCC) is the main type of liver tumor induced by kras(V12) expression. Moreover, mixed tumors with hepatocellular adenoma and hepatoblastoma (HB) were also frequently observed. Molecular analyses also indicated similar increase of phosphorylated ERK1/2 in all types of liver tumors, but nuclear localization of ß-catenin, a sign of malignant transformation, was found only in HCC and HB. Taken together, our new transgenic system reported in this study allows transgenic kras(V12) expression specifically in the zebrafish liver only by a brief exposure of mifepristone to induce permanent genomic recombination mediated by the Cre-loxP system.

Oncogenic KRAS promotes malignant brain tumors in zebrafish.

BACKGROUND: Zebrafish have been used as a vertebrate model to study human cancers such as melanoma, rhabdomyosarcoma, liver cancer, and leukemia as well as for high-throughput screening of small molecules of therapeutic value. However, they are just emerging as a model for human brain tumors, which are among the most devastating and difficult to treat. In this study, we evaluated zebrafish as a brain tumor model by overexpressing a human version of oncogenic KRAS (KRAS(G12V)). METHODS: Using zebrafish cytokeratin 5 (krt5) and glial fibrillary acidic protein (gfap) gene promoters, we activated Ras signaling in the zebrafish central nervous system (CNS) through transient and stable transgenic overexpression. Immunohistochemical analyses were performed to identify activated pathways in the resulting brain tumors. The effects of the MEK inhibitor U0126 on oncogenic KRAS were evaluated. RESULTS: We demonstrated that transient transgenic expression of KRAS(G12V) in putative neural stem and/or progenitor cells induced brain tumorigenesis. When expressed under the control of the krt5 gene promoter, KRAS(G12V) induced brain tumors in ventricular zones (VZ) at low frequency. The majority of other tumors were composed mostly of spindle and epithelioid cells, reminiscent of malignant peripheral nerve sheath tumors (MPNSTs). In contrast, when expressed under the control of the gfap gene promoter, KRAS(G12V) induced brain tumors in both VZs and brain parenchyma at higher frequency. Immunohistochemical analyses indicated prominent activation of the canonical RAS-RAF-ERK pathway, variable activation of the mTOR pathway, but no activation of the PI3K-AKT pathway. In a krt5-derived stable and inducible transgenic line, expression of oncogenic KRAS resulted in skin hyperplasia, and the MEK inhibitor U0126 effectively suppressed this pro-proliferative effects. In a gfap-derived stable and inducible line, expression of oncogenic KRAS led to significantly increased mitotic index in the spinal cord. CONCLUSIONS: Our studies demonstrate that zebrafish could be explored to study cellular origins and molecular mechanisms of brain tumorigenesis and could also be used as a platform for studying human oncogene function and for discovering oncogenic RAS inhibitors.

Myc-induced liver tumors in transgenic zebrafish can regress in tp53 null mutation.

Hepatocellular carcinoma (HCC) is currently one of the top lethal cancers with an increasing trend. Deregulation of MYC in HCC is frequently detected and always correlated with poor prognosis. As the zebrafish genome contains two differentially expressed zebrafish myc orthologs, myca and mycb, it remains unclear about the oncogenicity of the two zebrafish myc genes. In the present study, we developed two transgenic zebrafish lines to over-express myca and mycb respectively in the liver using a mifepristone-inducible system and found that both myc genes were oncogenic. Moreover, the transgenic expression of myca in hepatocytes caused robust liver tumors with several distinct phenotypes of variable severity. ~5% of myca transgenic fish developing multinodular HCC with cirrhosis after 8 months of induced myca expression. Apoptosis was also observed with myca expression; introduction of homozygous tp53(-/-) mutation into the myca transgenic fish reduced apoptosis and accelerated tumor progression. The malignant status of hepatocytes was dependent on continued expression of myca; withdrawal of the mifepristone inducer resulted in a rapid regression of liver tumors, and the tumor regression occurred even in the tp53(-/-) mutation background. Thus, our data demonstrated the robust oncogenicity of zebrafish myca and the requirement of sustained Myc overexpression for maintenance of the liver tumor phenotype in this transgenic model. Furthermore, tumor regression is independent of the function of Tp53.

Nonlesions, misdiagnoses, missed diagnoses, and other interpretive challenges in fish histopathology studies: a guide for investigators, authors, reviewers, and readers.

Differentiating salient histopathologic changes from normal anatomic features or tissue artifacts can be decidedly challenging, especially for the novice fish pathologist. As a consequence, findings of questionable accuracy may be reported inadvertently, and the potential negative impacts of publishing inaccurate histopathologic interpretations are not always fully appreciated. The objectives of this article are to illustrate a number of specific morphologic findings in commonly examined fish tissues (e.g., gills, liver, kidney, and gonads) that are frequently either misdiagnosed or underdiagnosed, and to address related issues involving the interpretation of histopathologic data. To enhance the utility of this article as a guide, photomicrographs of normal and abnormal specimens are presented. General recommendations for generating and publishing results from histopathology studies are additionally provided. It is hoped that the furnished information will be a useful resource for manuscript generation, by helping authors, reviewers, and readers to critically assess fish histopathologic data.

Histopathologic alterations associated with global gene expression due to chronic dietary TCDD exposure in juvenile zebrafish.

The goal of this project was to investigate the effects and possible developmental disease implication of chronic dietary TCDD exposure on global gene expression anchored to histopathologic analysis in juvenile zebrafish by functional genomic, histopathologic and analytic chemistry methods. Specifically, juvenile zebrafish were fed Biodiet starter with TCDD added at 0, 0.1, 1, 10 and 100 ppb, and fish were sampled following 0, 7, 14, 28 and 42 d after initiation of the exposure. TCDD accumulated in a dose- and time-dependent manner and 100 ppb TCDD caused TCDD accumulation in female (15.49 ppb) and male (18.04 ppb) fish at 28 d post exposure. Dietary TCDD caused multiple lesions in liver, kidney, intestine and ovary of zebrafish and functional dysregulation such as depletion of glycogen in liver, retrobulbar edema, degeneration of nasal neurosensory epithelium, underdevelopment of intestine, and diminution in the fraction of ovarian follicles containing vitellogenic oocytes. Importantly, lesions in nasal epithelium and evidence of endocrine disruption based on alternatively spliced vasa transcripts are two novel and significant results of this study. Microarray gene expression analysis comparing vehicle control to dietary TCDD revealed dysregulated genes involved in pathways associated with cardiac necrosis/cell death, cardiac fibrosis, renal necrosis/cell death and liver necrosis/cell death. These baseline toxicological effects provide evidence for the potential mechanisms of developmental dysfunctions induced by TCDD and vasa as a biomarker for ovarian developmental disruption.

Gene expression and pathologic alterations in juvenile rainbow trout due to chronic dietary TCDD exposure.

The goal of this project was to use functional genomic methods to identify molecular biomarkers as indicators of the impact of TCDD exposure in rainbow trout. Specifically, we investigated the effects of chronic dietary TCDD exposure on whole juvenile rainbow trout global gene expression associated with histopathological analysis. Juvenile rainbow trout were fed Biodiet starter with TCDD added at 0, 0.1, 1, 10 and 100 ppb (ngTCDD/g food), and fish were sampled from each group at 7, 14, 28 and 42 days after initiation of feeding. 100 ppb TCDD caused 100% mortality at 39 days. Fish fed with 100 ppb TCDD food had TCDD accumulation of 47.37 ppb (ngTCDD/g fish) in whole fish at 28 days. Histological analysis from TCDD-treated trout sampled from 28 and 42 days revealed that obvious lesions were found in skin, oropharynx, liver, gas bladder, intestine, pancreas, nose and kidney. In addition, TCDD caused anemia in peripheral blood, decreases in abdominal fat, increases of remodeling of fin rays, edema in pericardium and retrobulbar hemorrhage in the 100 ppb TCDD-treated rainbow trout compared to the control group at 28 days. Dose- and time-dependent global gene expression analyses were performed using the cGRASP 16,000 (16K) cDNA microarray. TCDD-responsive whole body transcripts identified in the microarray experiments have putative functions involved in various biological processes including growth, cell proliferation, metabolic process, and immune system processes. Nine microarray-identified genes were selected for QPCR validation. CYP1A3 and CYP1A1 were common up-regulated genes and HBB1 was a common down-regulated gene among each group based on microarray data, and their QPCR validations are consistent with microarray data for the 10 and 100 ppb TCDD treatment groups after 28 days exposure (p<0.05). In addition, in the 100 ppb group at 28 days, expression of complement component C3-1 and trypsin-1 precursor have a more than 10-fold induction from the microarray experiments, and their QPCR validations are consistent and showed significant induction in the 100 ppb group at 28 days (p<0.05). Overall, lesion in nasal epithelium is a novel and significant result in this study, and TCDD-responsive rainbow trout transcripts identified in the present study may lead to the development of new molecular biomarkers for assessing the potential impacts of environmental TCDD on rainbow trout populations.

A transgenic zebrafish liver tumor model with inducible Myc expression reveals conserved Myc signatures with mammalian liver tumors.

Myc is a pleiotropic transcription factor that is involved in many cellular activities relevant to carcinogenesis, including hepatocarcinogenesis. The zebrafish has been increasingly used to model human diseases and it is particularly valuable in helping to identify common and conserved molecular mechanisms in vertebrates. Here we generated a liver tumor model in transgenic zebrafish by liver-specific expression of mouse Myc using a Tet-On system. Dosage-dependent induction of Myc expression specifically in the liver was observed in our Myc transgenic zebrafish, TO(Myc), and the elevated Myc expression caused liver hyperplasia, which progressed to hepatocellular adenoma and carcinoma with prolonged induction. Next generation sequencing-based transcriptomic analyses indicated that ribosome proteins were overwhelmingly upregulated in the Myc-induced liver tumors. Cross-species analyses showed that the zebrafish Myc model correlated well with Myc transgenic mouse models for liver cancers. The Myc-induced zebrafish liver tumors also possessed molecular signatures highly similar to human those of hepatocellular carcinoma. Finally, we found that a small Myc target gene set of 16 genes could be used to identify liver tumors due to Myc upregulation. Thus, our zebrafish model demonstrated the conserved role of Myc in promoting hepatocarcinogenesis in all vertebrate species.

An inducible kras(V12) transgenic zebrafish model for liver tumorigenesis and chemical drug screening.

Because Ras signaling is frequently activated by major hepatocellular carcinoma etiological factors, a transgenic zebrafish constitutively expressing the kras(V12) oncogene in the liver was previously generated by our laboratory. Although this model depicted and uncovered the conservation between zebrafish and human liver tumorigenesis, the low tumor incidence and early mortality limit its use for further studies of tumor progression and inhibition. Here, we employed a mifepristone-inducible transgenic system to achieve inducible kras(V12) expression in the liver. The system consisted of two transgenic lines: the liver-driver line had a liver-specific fabp10 promoter to produce the LexPR chimeric transactivator, and the Ras-effector line contained a LexA-binding site to control EGFP-kras(V12) expression. In double-transgenic zebrafish (driver-effector) embryos and adults, we demonstrated mifepristone-inducible EGFP-kras(V12) expression in the liver. Robust and homogeneous liver tumors developed in 100% of double-transgenic fish after 1 month of induction and the tumors progressed from hyperplasia by 1 week post-treatment (wpt) to carcinoma by 4 wpt. Strikingly, liver tumorigenesis was found to be 'addicted' to Ras signaling for tumor maintenance, because mifepristone withdrawal led to tumor regression via cell death in transgenic fish. We further demonstrated the potential use of the transparent EGFP-kras(V12) larvae in inhibitor treatments to suppress Ras-driven liver tumorigenesis by targeting its downstream effectors, including the Raf-MEK-ERK and PI3K-AKT-mTOR pathways. Collectively, this mifepristone-inducible and reversible kras(V12) transgenic system offers a novel model for understanding hepatocarcinogenesis and a high-throughput screening platform for anti-cancer drugs.

Neoplasia and neoplasm-associated lesions in laboratory colonies of zebrafish emphasizing key influences of diet and aquaculture system design.

During the past decade, the zebrafish has emerged as a leading model for mechanistic cancer research because of its sophisticated genetic and genomic resources, its tractability for tissue targeting of transgene expression, its efficiency for forward genetic approaches to cancer model development, and its cost effectiveness for enhancer and suppressor screens once a cancer model is established. However, in contrast with other laboratory animal species widely used as cancer models, much basic cancer biology information is lacking in zebrafish. As yet, data are not published regarding dietary influences on neoplasm incidences in zebrafish. Little information is available regarding spontaneous tumor incidences or histologic types in wild-type lines of zebrafish. So far, a comprehensive database documenting the full spectrum of neoplasia in various organ systems and tissues is not available for zebrafish as it is for other intensely studied laboratory animal species. This article confirms that, as in other species, diet and husbandry can profoundly influence tumor incidences and histologic spectra in zebrafish. We show that in many laboratory colonies wild-type lines of zebrafish exhibit elevated neoplasm incidences and neoplasm-associated lesions such as heptocyte megalocytosis. We present experimental evidence showing that certain diet and water management regimens can result in high incidences of neoplasia and neoplasm-associated lesions. We document the wide array of benign and malignant neoplasms affecting nearly every organ, tissue, and cell type in zebrafish, in some cases as a spontaneous aging change, and in other cases due to carcinogen treatment or genetic manipulation.

Inducible and repressable oncogene-addicted hepatocellular carcinoma in Tet-on xmrk transgenic zebrafish.

BACKGROUND & AIMS: Liver cancer, mainly hepatocellular carcinoma, is a major malignancy and currently there are no effective treatment protocols due to insufficient understanding of hepatocarcinogenesis. As a potentially high-throughput and cost-effective experimental model, the zebrafish is increasingly recognized for disease studies. Here, we aim at using the zebrafish to generate a convenient hepatocellular carcinoma model. METHODS: Using the Tet-on system for liver-specific expression of fish oncogene xmrk, a hyperactive version of epidermal growth factor receptor homolog, we have generated transgenic zebrafish with inducible development of liver cancer. RESULTS: Liver tumors were rapidly induced with 100% penetrance in both juvenile and adult xmrk transgenic fish. Histological examination indicated that they all showed features of hepatocellular carcinoma. The induced liver tumors regressed rapidly upon inducer withdrawal. During the tumor induction stage, we detected increased cell proliferation and activation of Xmrk downstream targets Erk and Stat5, which were important for liver tumorigenesis as proved by inhibition experiments. When tumors regressed, there were decreased phosphorylated Erk and Stat5 accompanied with an increase in apoptosis. CONCLUSIONS: Our zebrafish model demonstrates the potential of a hyperactivated epidermal growth factor receptor pathway in initiating heptocarcinogenesis. It provides clear evidence for the requirement of only a single oncogene for HCC initiation and maintenance and is thus a convenient model for further investigation of oncogene addiction and future anti-cancer drug screening.

Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections.

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation, or small spores in nuclei (i.e. Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores is recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells, and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fite's acid fast, Giemsa, and H&E stains on 8 aquatic microsporidian organisms that were readily available in our 2 laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum, and an unidentified microsporidian from UK mitten crabs Eriocheir sinensis. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the 2 microsporidia from invertebrates: S. mytilovum and the unidentified microsporidian from E. sinensis.

A high level of liver-specific expression of oncogenic Kras(V12) drives robust liver tumorigenesis in transgenic zebrafish.

Human liver cancer is one of the deadliest cancers worldwide, with hepatocellular carcinoma (HCC) being the most common type. Aberrant Ras signaling has been implicated in the development and progression of human HCC, but a complete understanding of the molecular mechanisms of this protein in hepatocarcinogenesis remains elusive. In this study, a stable in vivo liver cancer model using transgenic zebrafish was generated to elucidate Ras-driven tumorigenesis in HCC. Using the liver-specific fabp10 (fatty acid binding protein 10) promoter, we overexpressed oncogenic kras(V12) specifically in the transgenic zebrafish liver. Only a high level of kras(V12) expression initiated liver tumorigenesis, which progressed from hyperplasia to benign and malignant tumors with activation of the Ras-Raf-MEK-ERK and Wnt-ß-catenin pathways. Histological diagnosis of zebrafish tumors identified HCC as the main lesion. The tumors were invasive and transplantable, indicating malignancy of these HCC cells. Oncogenic kras(V12) was also found to trigger p53-dependent senescence as a tumor suppressive barrier in the pre-neoplastic stage. Microarray analysis of zebrafish liver hyperplasia and HCC uncovered the deregulation of several stage-specific and common biological processes and signaling pathways responsible for kras(V12)-driven liver tumorigenesis that recapitulated the molecular hallmarks of human liver cancer. Cross-species comparisons of cancer transcriptomes further defined a HCC-specific gene signature as well as a liver cancer progression gene signature that are evolutionarily conserved between human and zebrafish. Collectively, our study presents a comprehensive portrait of molecular mechanisms during progressive Ras-induced HCC. These observations indicate the validity of our transgenic zebrafish to model human liver cancer, and this model might act as a useful platform for drug screening and identifying new therapeutic targets.

Normal anatomy and histology of the adult zebrafish.

The zebrafish has been shown to be an excellent vertebrate model for studying the roles of specific genes and signaling pathways. The sequencing of its genome and the relative ease with which gene modifications can be performed have led to the creation of numerous human disease models that can be used for testing the potential and the toxicity of new pharmaceutical compounds. Many pharmaceutical companies already use the zebrafish for prescreening purposes. So far, the focus has been on ecotoxicity and the effects on embryonic development, but there is a trend to expand the use of the zebrafish with acute, subchronic, and chronic toxicity studies that are currently still carried out with the more conventional test animals such as rodents. However, before we can fully realize the potential of the zebrafish as an animal model for understanding human development, disease, and toxicology, we must first greatly advance our knowledge of normal zebrafish physiology, anatomy, and histology. To further this knowledge, we describe, in the present article, location and histology of the major zebrafish organ systems with a brief description of their function.

Eosinophils in the zebrafish: prospective isolation, characterization, and eosinophilia induction by helminth determinants.

Eosinophils are granulocytic leukocytes implicated in numerous aspects of immunity and disease. The precise functions of eosinophils, however, remain enigmatic. Alternative models to study eosinophil biology may thus yield novel insights into their function. Eosinophilic cells have been observed in zebrafish but have not been thoroughly characterized. We used a gata2:eGFP transgenic animal to enable prospective isolation and characterization of zebrafish eosinophils, and demonstrate that all gata2(hi) cells in adult hematopoietic tissues are eosinophils. Although eosinophils are rare in most organs, they are readily isolated from whole kidney marrow and abundant within the peritoneal cavity. Molecular analyses demonstrate that zebrafish eosinophils express genes important for the activities of mammalian eosinophils. In addition, gata2(hi) cells degranulate in response to helminth extract. Chronic exposure to helminth- related allergens resulted in profound eosinophilia, demonstrating that eosinophil responses to allergens have been conserved over evolution. Importantly, infection of adult zebrafish with Pseudocapillaria tomentosa, a natural nematode pathogen of teleosts, caused marked increases in eosinophil number within the intestine. Together, these observations support a conserved role for eosinophils in the response to helminth antigens or infection and provide a new model to better understand how parasitic worms activate, co-opt, or evade the vertebrate immune response.

Transgenic expression of walleye dermal sarcoma virus rv-cyclin gene in zebrafish and its suppressive effect on liver tumor development after carcinogen treatment.

A retrovirus homologue gene of cellular cyclin D1, walleye dermal sarcoma virus rv-cyclin gene (orf A or rv-cyclin), was expressed in the livers of zebrafish under the control of liver fatty acid-binding protein (lfabp) promoter. To prevent possible fatality caused by overexpression of the oncogene, the GAL4/upstream activation sequence (GAL4/UAS) system was used to maintain the transgenic lines. Thus, both GAL4-activator [Tg(lfabp:GAL4)] and UAS-effector [Tg(UAS:rvcyclin)] lines were generated, and the rv-cyclin gene was activated in the liver after crossing these two lines. Since no obvious neoplasia phenotypes were observed in the double-transgenic line, cancer susceptibility of the transgenic fish expressing rv-cyclin was tested by carcinogen treatment. Unexpectedly, transgenic fish expressing rv-cyclin gene (rvcyclin+) were more resistant to the carcinogen than siblings not expressing this gene (rvcyclin-). Lower incidences of multiple and malignant liver tumors were observed in rvcyclin+ than in rvcyclin- fish, and the liver tumors in the rvcyclin+ group appeared later and were less malignant. These results suggest that expression of rv-cyclin protects the fish liver from carcinogen damage and delays onset of malignancy. These findings indicate that transgenic fish models are powerful systems for investigating mechanisms of inhibition and regression of liver tumors.

Nonlinear cancer response at ultralow dose: a 40800-animal ED(001) tumor and biomarker study.

Assessment of human cancer risk from animal carcinogen studies is severely limited by inadequate experimental data at environmentally relevant exposures and by procedures requiring modeled extrapolations many orders of magnitude below observable data. We used rainbow trout, an animal model well-suited to ultralow-dose carcinogenesis research, to explore dose-response down to a targeted 10 excess liver tumors per 10000 animals (ED(001)). A total of 40800 trout were fed 0-225 ppm dibenzo[a,l]pyrene (DBP) for 4 weeks, sampled for biomarker analyses, and returned to control diet for 9 months prior to gross and histologic examination. Suspect tumors were confirmed by pathology, and resulting incidences were modeled and compared to the default EPA LED(10) linear extrapolation method. The study provided observed incidence data down to two above-background liver tumors per 10000 animals at the lowest dose (that is, an unmodeled ED(0002) measurement). Among nine statistical models explored, three were determined to fit the liver data well-linear probit, quadratic logit, and Ryzin-Rai. None of these fitted models is compatible with the LED(10) default assumption, and all fell increasingly below the default extrapolation with decreasing DBP dose. Low-dose tumor response was also not predictable from hepatic DBP-DNA adduct biomarkers, which accumulated as a power function of dose (adducts = 100 x DBP(1.31)). Two-order extrapolations below the modeled tumor data predicted DBP doses producing one excess cancer per million individuals (ED(10)(-6)) that were 500-1500-fold higher than that predicted by the five-order LED(10) extrapolation. These results are considered specific to the animal model, carcinogen, and protocol used. They provide the first experimental estimation in any model of the degree of conservatism that may exist for the EPA default linear assumption for a genotoxic carcinogen.