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Architectural pattern, tissue and cellular morphology in livers of fishes: Relationship to experimentally-induced neoplastic responses
Abstract
The teleost liver is one of the most sensitive organs to show alteration in biochemistry, physiology and structure following exposure to various types of environmental pollutants. Despite the importance of this organ to environmental toxicology and to ecotoxicology where biomarkers of exposure and of deleterious effect are found, the architectural pattern is not well known. This chapter reviews an architectural plan for teleost liver and compares that to the often cited mammalian pattern. Hepatic tubules composed principally of hepatocytes and biliary epithelial cells are in close proximity to lacunae which are of mesodermal origin. As is described, the tubule and lacunae concepts provide a means to better interpret morphologic alterations following exposure. These concepts are used to illustrate features of the chronic toxicity following exposure to proven carcinogens.
... Their small size allows for whole-body histologic sectioning in the parasagittal plane for analysis of multiple organs and tissues on a few slides. The single lobed liver of medaka enables clean removal of the entire organ and avoids issues of hepatic lobar distribution and size of focal alterations (Hinton and Couch 1998). Additionally, toxic effects on the liver that alter the structure and function of cells and tissues have been well-described in this model (Evans 1987;Hinton et al. 2001). ...
... Fish lack the fixed Kupffer cells seen in mammalian sinusoids (Hinton et al. 2008). Rather, they form macrophage aggregates in perisinusoidal spaces (Hinton and Couch 1998;Hinton et al. 2008). While we observed macrophage aggregates in sections of livers of exposed individuals, the techniques used in our study did not provide evidence to indicate that macrophages were a specific target of any of the materials tested. ...
... It is well-known that hepatic biotransformation is a detoxification process important for the excretion of toxic substances (Braunbeck 1998). Both hepatocytes and intrahepatic biliary epithelial cells are intimately involved (Hinton and Couch 1998). Uptake of substances at the sinusoidal surface of hepatocytes followed by their intracellular transport and later excretion into bile fluid at bile canaliculi are essential functions in detoxification (Hinton et al. 2001). ...
Polymer nanocomposites combine the versatile, lightweight characteristics of polymers with the properties of nanomaterials. Polyethylene terephthalate glycol (PETG) is commonly used in polymer additive manufacturing due to its controllable transparency, high modulus, and mechanical properties. Multi-walled carbon nanotubes (MWCNTs) add tensile strength, electrical conductivity, and thermal stability. The increased use of nanocomposites has led to concern over potential human health risks. We assessed morphologic alterations to determine impacts of ingested abraded nanocomposites compared to its component materials, pristine MWCNTs (1000 mg/L) and PETG. Adult transparent Japanese medaka (Oryzias latipes) were administered materials via oral gavage in 7 doses over 16 days. In vivo observations revealed altered livers and gallbladders following exposure to pristine MWCNTs and nanocomposites. Subsequent histologic sections showed fish exposed to pristine MWCNTs had highly altered biliary structures, and exposure to nanocomposites resulted in hepatocellular alteration. Thyroid follicle proliferation was also observed in fish exposed to materials containing MWCNTs. Transmission electron microscopy of livers showed that hepatocytes of fish exposed to MWCNTs had widespread swelling of rough endoplasmic reticulum, pronounced lysosomal activity, and swelling of intrahepatic biliary passageways. Fish exposed to nanocomposites had areas of degenerated hepatocytes with interspersed cellular debris. Each analysis showed that fish exposed to pristine PETG were most similar to controls. These results suggest that MWCNTs are the source of toxicity in abraded nanocomposite materials but that nanocomposites may also have some unique effects. The similarities of many teleost and mammalian tissues are such that these findings may indicate human health risks.
... Вместо портальной архитектуры гепатоциты в печени рыб расположены в виде канальцев с желчными протоками, проходящими между 2 рядами гепатоцитов. Апикальные мембраны обращены к внутренней части канальца, а синусоиды располагаются на базальной стороне гепатоцитов [11,12]. Имеется 2 типа желчных клеток: маленькие предлежащие желчные эпителиальные клетки, которые создают внутриклеточные просветы для транспортировки желчи из гепатоцитов, и более крупные столбчатые холангиоциты, которые образуют полноценную внутрипеченочную желчевыводящую систему. ...
... Имеется 2 типа желчных клеток: маленькие предлежащие желчные эпителиальные клетки, которые создают внутриклеточные просветы для транспортировки желчи из гепатоцитов, и более крупные столбчатые холангиоциты, которые образуют полноценную внутрипеченочную желчевыводящую систему. Сравнительно недавно были описаны звездчатые клетки печени (миофибробласты, которые активируются и выделяют внеклеточный матрикс при повреждении печени [11,12]. Хотя патогенетическая основа и сигнальные пути заболеваний печени у млекопитающих и рыб, по-видимому, схожа, различия в микросреде могут способствовать специфическим для вида реакциям на повреждение клеток [13,14]. ...
This article describes the morphological structure of the zebrafish liver and its comparison with the organization of the liver of mammals, known methods for inducing liver pathologies in these fish, and assesses the prospects for using Danio rerio (Zebrafish) as biological models in the context of studying hepatotoxic effects.
The scientific novelty of the publication lies in the complexity of the ongoing review of existing studies in the field of using model animals to assess various hepatotoxic effects, identifying the most promising approaches from the point of view of veterinary hepatology for their further possible introduction into industry practice.
The information retrieval methodology was based on such general scientific methods of cognition as: a review of specialized search engines and databases of scientific and research data (Scopus, WoS, PubMed) over the past 10 years, of which the most informative ones were selected, analysis of the identified results, their comparison by relevance.
Summing up the results of the search, we can conclude that the results of relevant experiments indicate the promise of studying hepatotoxic effects on zebrafish, which represent a promising and cost-effective alternative to other model objects. Danio rerio can reproduce in detail and with high accuracy the patterns of occurrence, development and outcome of pathological processes in mammalian organs due to the high level of genetic preservation and similar morphology and functions of organs. It is believed that further technical development and characterization of these biomodels in the context of the study of hepatotoxicity will provide new insights into the molecular and cellular mechanisms underlying disease pathogenesis, thereby providing multiple opportunities for the identification and validation of new therapeutic targets and treatments.
... The zebrafish liver is organized in 3 contiguous lobes (2 lateral and 1 ventral) and these lobes lack the pedicle that separates the distinct lobes in the mammalian liver. Instead of a portal architecture, hepatocytes in fish livers are arranged in tubules, with bile ductules coursing between 2 rows of hepatocytes [16][17][18][19] (Figure 2B and C). The apical membranes face the inside of the tubule, and sinusoids track the basal side of hepatocytes ( Figure 2B). ...
... Fish have 2 types of biliary cells: small preductal biliary epithelial cells, which create intracellular lumens for bile transport from the hepatocytes, and larger columnar cholangiocytes, which form the full intrahepatic biliary system. [16][17][18][19] Hepatic stellate cells were recently described in zebrafish as myofibroblasts that become activated and secrete extracellular matrix on hepatic injury. 27 Endothelial cells line the hepatic vessels, with sinusoids at the basal side of hepatocytes. ...
As the incidence of hepatobiliary diseases increases, we must improve our understanding of the molecular, cellular, and physiological factors that contribute to liver disease pathogenesis. Animal models help us identify disease mechanisms that might be targeted therapeutically. Zebrafish (Danio rerio) have traditionally been used to study embryonic development, but are also important to the study of liver disease. Zebrafish embryos develop rapidly-all of their digestive organs are mature in larvae by 5 days of age. At this stage, they can develop hepatobiliary diseases caused by developmental defects or toxin- or ethanol-induced injury, and manifest premalignant changes within weeks. Zebrafish are similar to humans in hepatic cellular composition, function, signaling, and response to injury, as well as the cellular processes that mediate liver diseases. Genes are highly conserved between humans and zebrafish, making them a useful system to study the basic mechanisms of liver disease. We can perform genetic screens to identify novel genes involved in specific disease processes, and chemical screens to identify pathways and compounds that act on specific processes. We review how studies of zebrafish have advanced our understanding of inherited and acquired liver diseases, as well as liver cancer and regeneration.
Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.
... Importantly gills facilitate gaseous exchange across extremely thin diffusion distances between the external water and internal blood, with particular sensitivity to contamination Heath, 1995;Wood, 2001). Additionally, liver is also recognised as the main detoxification organ which is highly sensitive to toxicant exposure, and generally accumulates contaminants to higher concentrations than other tissues (Heath, 1995;Hinton and Couch, 1998;Hinton et al., 2001). It is therefore not surprising that both the gills and liver exhibited the highest severity of alteration due to their level of exposure and sensitivity to contaminants. ...
... In contrast to gills, the liver is not directly exposed to water born contaminants, which may account for the less pronounced severity of alteration in liver. However, the liver is representative of uptake over time and indicates the cumulative response to wastewater exposure relative to the gill based on the detoxification function of the liver (Heath, 1995;Hinton and Couch, 1998;Hinton et al., 2001). ...
A quantitative Histological Health Index (HHI) was applied to Antarctic rock cod (Trematomus bernacchii) using gill, liver, spleen, kidney and gonad to assess the impact of wastewater effluent from Davis Station, East Antarctica. A total of 120 fish were collected from 6 sites in the Prydz Bay region of East Antarctica at varying distances from the wastewater outfall. The HHI revealed a greater severity of alteration in fish at the wastewater outfall, which decreased stepwise with distance. Gill and liver displayed the greatest severity of alteration in fish occurring in close proximity to the wastewater outfall, showing severe and pronounced alteration respectively. Findings of the HHI add to a growing weight of evidence indicating that the current level of wastewater treatment at Davis Station is insufficient to prevent impact to the surrounding environment. The HHI for T. bernacchii developed in this study is recommended as a useful risk assessment tool for assessing in situ, sub-lethal impacts from station-derived contamination in coastal regions throughout Antarctica.
Copyright © 2015 Elsevier Ltd. All rights reserved.
... The structure of the vertebrate liver has been studied for a long time, been kept two main models: the first one, representative of mammalian liver, is called lobular model, and the second one, related to basal vertebrate liver, is reported as a tubular model (Yao et al. 2012). The tubular model is characterized by observation, in longitudinal section, of two rows of hepatocytes forming a tubular lumen or biliary passageway in their apical membranes, whereas basal membranes keep bordering the sinusoidal space (Hinton and Couch 1998). Previous researches in fish liver have established that there is a great variety in the structure of the organ in different species (Schar et al. 1985; Robertson and Bradley 1992). ...
... There are also obvious differences even in the same fish family, so it is not possible to describe the architecture of the liver of teleosts in a generalized way (Rocha and Monteiro 1999; Akiyoshi and Inoue 2004; Flores-Lopes and Malabarba 2007). Thus, the knowledge of such particularities is essential for the correct identification of the hepatic structure in different species, and for understanding the structural changes and damages caused by water pollutants (Hinton and Couch 1998; Figueiredo-Fernandes et al. 2007). The species Astyanax altiparanae belongs to the family Characidae, and the genus Astyanax is the most common and diverse (Garutti and Britski 1997; Fernandes and Martins-Santos 2004). ...
Studies on the morphology of the liver of teleosts reflect some controversy in the interpretation of the data, but also provide confirmation of variations in the structure of the organ in several species. Thus, we intend to understand the specific structural organization of the liver of Astyanax altiparanae. Specimens were collected in the city of Andirá, Paraná, Brazil. The livers were processed according to histological routine for inclusion in Paraplast, and the sections were stained with HE and Mallory's trichrome or followed the protocol for fluorescence immunohistochemistry, anti-cytokeratin. The liver of A. altiparanae was covered by a capsule of connective tissue, without delimiting lobes. The hepatocytes had an arrangement in cords around sinusoids. Melanomacrophage centers were observed. The vascular components and intrahepatic pancreatic acini were distributed between hepatocytes. Presence of cytokeratin was detected in tissues that lined the liver and endothelial cells of sinusoids. The comparison of the liver of A. altiparanae to other characids corroborates with the fact that there is variation in the morphology of the liver even between closely related species. Moreover, it appears that in this species, endothelial cells of sinusoids can synthesize the cytokeratin filaments required for the regulation of blood flow in capillaries in adults.
... The liver has a vital physiological role and its functional diversity and complexity are credited to be rivalled only by the central nervous system (Jones and Spring-Mills 1988). Despite great basic differences as to how hepatocytes are three-dimensionally arranged (Hinton and Couch 1998;Hardman et al. 2007), from fishes to mammals they are the main structural component of the liver parenchyma. In the relatively few species studied, hepatocytes make up around 80% of the liver volume in both fishes and mammals (e.g., Weibel et al. 1969;Rocha et al. 1997). ...
... The fish liver has been a constant focus of attention in research. The interest in knowing how liver structure differs in vertebrates is an old driving force, and the piscine liver in particular has always been a matter of debate and renditions (Elias and Bengelsdorf 1952;Beresford and Henninger 1986;Hinton and Couch 1998). Another point of interest is that fish liver is prone to tumors (Nigrelli and Jakowska 1961); therefore, it is being used as a model in the study of carcinogenesis, namely that related with pollutants (Bailey et al. 1996). ...
A firm knowledge of the normal structure is crucial for evaluating pathological processes and morphofunctional correlations.
Stereological liver structure characterization had its debut for mammals in the 1960s, but only in the 1980s did it start
to be used in fishes. Using stereology, our aim was to verify the hypothesis that in parallel with the well-known annual seasonal
changes in the liver–body ratio of brown trout, hepatocytes would vary their number and/or size, and that gender differences
likely exist. Three-year-old specimens were used. Five animals per gender were examined in May (endogenous vitellogenesis),
September (exogenous vitellogenesis), and February (spawning season end). The liver was fixed by perfusion, and its total
volume estimated. Systematically sampled material was embedded in epoxy or in metachrylate resins. Stereology was executed
on light and electron microscopy images. Unbiased design-based techniques were applied, using physical disectors and differential
point counting. Target parameters were the relative (per unit volume) and total number of hepatocytes, the mean cell and nuclear
volumes, and the total volumes of hepatocytes and their nuclei. Data support that in both genders the number of hepatocytes
and the volume of its nucleus change along the breeding cycle. The cell number increased from endogenous to exogenous vitellogenesis
(accompanying relative liver size gains), later followed by a decline in the cell number, still detectable after the spawning
season. The total liver volumes of the cell and nucleus also increased from May to September in females, despite that the
mean hepatocyte nuclear volume showed a minimum in September. No statistical changes in the mean cell volume were detected,
regardless of the tendency for lower mean values in September. Changes were more marked in females and showed a higher correlation
with the gonad weight. It was firstly suggested that numerical (rather than cell size) changes govern the shifts of the relative
liver weight seen during the brown trout annual breeding cycle, and eventually of other fishes. We hypothesized that there
are seasonal cycles of hepatocyte mitosis (from after spawning to exogenous vitellogenesis) and of apoptosis (at spawning).
These cycles would be regulated by sex steroids, being more striking in females.
... Together these studies suggest that CYP1s are having a role in tumor initiation and toxic metabolite formation but additional in vivo studies and isoform specific CYP1 inhibitors are needed. The structural anatomy and essential functions of the fish liver associated with chemically induced toxicity, as well as the comparisons with mammalian liver physiology, have been well described (Hampton et al., 1985(Hampton et al., , 1988Hinton and Couch, 1998). These fundamental studies have provided a strong background for investigating the role of CYP1C1 versus CYP1A in PAH-induced liver toxicity. ...
... The biological significance of CYP1C1 expression in cholangiocytes, but not hepatocytes is unknown, although it has been proposed that a stem cell in the liver could be activated following hepatocarcinogen exposure and liver injury (Sell, 1990). In fish, biliary preductular epithelial cells could be the stem cell counterparts to rodent oval cells (Hinton and Couch, 1998). This hypothesis could explain why CYP1C1 was only detected in proliferated biliary epithelial cells and not the hepatocyte-derived neoplasms. ...
CYP1C1 is a relatively newly identified member of the cytochrome P450 family 1 in teleost fish. However, CYP1C1's expression and physiological roles relative to the more recognized CYP1A in polycyclic aromatic hydrocarbons (PAHs) induced toxicities are unclear. Fundulus heteroclitus fry were exposed at 6-8 days post-hatch (dph) and again at 13-15dph for 6h to dimethyl sulfoxide (DMSO) control, 5mg/L benzo[a]pyrene (BaP), or 5mg/L dimethylbenzanthracene (DMBA). Fry were euthanized at 0, 6, 18, 24 and 30h after the second exposure. In these groups, both CYP1A and CYP1C1 protein expression were induced within 6h after the second exposure. Immunohistochemistry (IHC) results from fry revealed strongest CYP1C1 expression in renal tubular and intestinal epithelial cells. Additional fish were examined for liver lesions 8 months after initial exposure. Gross lesions were observed in 20% of the BaP and 35% of the DMBA-treated fish livers. Histopathologic findings included foci of cellular alteration and neoplasms, including hepatocellular adenoma, hepatocellular carcinoma and cholangioma. Strong CYP1A immunostaining was detected diffusely in altered cell foci and on the invading margin of hepatocelluar carcinomas. Lower CYP1A expression was seen in central regions of the neoplasms. In contrast, CYP1C1 was only detectable and highly expressed in proliferated bile duct epithelial cells. Our CYP1C1 results suggest the potential for tissue specific CYP1C1-mediated PAH metabolism but not a more chronic role in progression to liver hepatocellular carcinoma.
... The fish liver, similar to mammals, is an exocrine and endocrine gland that acts (1) on nutrient processing and storage, (2) on the synthesis of enzymes and other proteins, (3) in bile secretion as well in (4) metabolism of xenobiotic compounds (Wolf and Wheele 2018). Further, the liver is one of the most sensitive organs that exhibits biochemical, physiological, and structural alterations following exposure to contaminants (Arman and Clarke 2021;Hinton and Couch 1998;Huguet et al. 2019;Zurawell et al. 2005). Although the liver is one of the main CYN-target organs (Buratti et al. 2017;Guzmán-Guillén et al. 2014;Rabelo et al. 2021), few investigators CYN-mediated toxicity and bioaccumulation in the liver of Neotropical fish species (da Rabelo et al. 2021), especially utilizing multiple biomarkers of exposure. ...
... In recent years, zebrafish models have been widely used to evaluate liver toxicity (Zhang et al., 2017a;Huo et al., 2019). The structure, cellular composition, main physiological processes, function, and response to injury of zebrafish liver are found to be similar to those of the human liver (Hinton and Couch, 1998;Field et al., 2003;Goessling and Sadler, 2015). Zebrafish larvae are virtually transparent, enabling the development and the morphological changes in liver visible (Hill et al., 2012). ...
Impurities in pharmaceuticals of potentially hazardous materials may cause drug safety problems. Macrolide antibiotic preparations include active pharmaceutical ingredients (APIs) and different types of impurities with similar structures, and the amount of these impurities is usually very low and difficult to be separated for toxicity evaluation. Our previous study indicated that hepatotoxicity induced by macrolides was correlated with c-fos overexpression. Here, we report an assessment of macrolide-related liver toxicity by ADMET prediction, molecular docking, structure–toxicity relationship, and experimental verification via detection of the c-fos gene expression in liver cells. The results showed that a rapid assessment model for the prediction of hepatotoxicity of macrolide antibiotics could be established by calculation of the -CDOCKER interaction energy score with the FosB/JunD bZIP domain and then confirmed by the detection of the c-fos gene expression in L02 cells. Telithromycin, a positive compound of liver toxicity, was used to verify the correctness of the model through comparative analysis of liver toxicity in zebrafish and cytotoxicity in L02 cells exposed to telithromycin and azithromycin. The prediction interval (48.1∼53.1) for quantitative hepatotoxicity in the model was calculated from the docking scores of seven macrolide antibiotics commonly used in clinics. We performed the prediction interval to virtual screening of azithromycin impurities with high hepatotoxicity and then experimentally confirmed by liver toxicity in zebrafish and c-fos gene expression. Simultaneously, we found the hepatotoxicity of azithromycin impurities may be related to the charge of nitrogen (N) atoms on the side chain group at the C5 position via structure–toxicity relationship of azithromycin impurities with different structures. This study provides a theoretical basis for improvement of the quality of macrolide antibiotics.
... In addition to mice, zebrafish models of ALGS have also been described. The extrahepatic and intrahepatic biliary system has been well-characterized in these animals (59)(60)(61), and both morpholino and transgenic perturbation of jagged genes has been successful in replicating the spectrum of clinical features associated with the disease (60,62). Zebrafish express three jagged genes (jag1a, jag1b, and jag2b) and targeted morpholino knockdown of both jag1b and jag2b has been shown to phenocopy ALGS, with fish displaying liver, kidney, pancreatic, craniofacial, and cardiac defects (60). ...
The observation of bile duct paucity is an important diagnostic finding in children, occurring in roughly 11% of pediatric liver biopsies. Alagille syndrome (ALGS) is a well-defined syndromic form of intrahepatic bile duct paucity that is accompanied by a number of other key features, including cardiac, facial, ocular, and vertebral abnormalities. In the absence of these additional clinical characteristics, intrahepatic bile duct paucity results in a broad differential diagnosis that requires supplementary testing and characterization. Nearly 30 years after ALGS was first described, genetic studies identified a causative gene, JAGGED1, which spearheaded over two decades of research aimed to meticulously delineate the molecular underpinnings of ALGS. These advancements have characterized ALGS as a genetic disease and led to testing strategies that offer the ability to detect a pathogenic genetic variant in almost 97% of individuals with ALGS. Having a molecular understanding of ALGS has allowed for the development of numerous in vitro and in vivo disease models, which have provided hope and promise for the future generation of gene-based and protein-based therapies. Generation of these disease models has offered scientists a mechanism to study the dynamics of bile duct development and regeneration, and in doing so, produced tools that are applicable to the understanding of other congenital and acquired liver diseases.
... Owing to their positive relationship with contaminant exposure, biliary preductular cell hyperplasias are considered as putative preneoplastic changes (Blazer et al, 2009b), but parasitic infections can also cause proliferations in fish bile ducts (Blazer et al., 2007). In contrast, basophilic cellular foci have been shown to transition into neoplasms in different fish species, such as rainbow trout Oncorhynchus mykiss (Hendricks et al., 1984), mosquitofish Gambusia affinis (Law et al., 1994), medaka Oryzias latipes, guppies Poecilia reticulate (Hawkins et al., 1988), and sheepshead minnow Cyprinodon variegatus (Hinton and Couch, 1998), and the same holds true for eosinophilic and vacuolated cellular foci in the presence of contaminants, like diethylnitrosamine and PAHs (Blazer et al., 2014). In general, foci of cellular alteration may also represent the early stages of cancer (Baumann and Okihiro, 2000). ...
Liver and skin tumours in brown bullhead (Ameiurus nebulosus) and white sucker (Catostomus commersoni) have been associated with contamination of the aquatic environment. In this study, we present a Bayesian hierarchical logistic-Bernoulli framework that considers the role of fish covariates (e.g., age, total weight, gonad weight, liver weight, fork length) on tumour prevalence to identify the presence of “hot-spots” around the Canadian waters of the Great Lakes, where high fish tumour frequencies are registered. We developed methods to discern the degree of impairment that are either based on the comparison of tumour frequencies in contaminated (or impacted) sites against those predicted in their corresponding reference areas, or the assessment of the prevailing conditions in impaired sites independently, without the need to establish baseline conditions for comparison purposes. Our modelling study predicts low frequencies of neoplastic tumours in all the impacted locations. In contrast, the same comparisons with the preneoplastic lesions provided evidence of distinct differences between impacted and reference sites in Jackfish Bay, St. Mary’s River, Niagara River, Hamilton Harbour, and Bay of Quinte. We also found weak to moderately strong relationships between tumour occurrence and fish physical characteristics that varied considerably in terms of their strength and nature (sign) among the different locations. Our study concludes that the prevalence of neoplastic tumours appears to have reached acceptable levels around the Great Lakes, but the distinctly higher levels of preneoplasms in several impacted locations underscore the need to improve our understanding of the lesions that may lead to carcinogenesis.
... Zebrafish (Danio rerio) is a new model organism, which has been widely used in liver diseases, including hyperlipidemia, ALD. All of their digestive organs are mature and functional in larvae by 72 dpf (Hinton and Couch, 1998). In our study, liver fluorescence of Transgenic (fabp10: EGFP) zebrafish reflected the morphology of the liver directly under a microscope, which indicated that liver morphology was damaged after modeling, while restored after quercetin treatment (Fig. 1A). ...
Ethnopharmacological relevanceQuercetin is the active component of the higher content in PCP, which exerts various biological activities such as anti-obesity effect, anti-inflammatory and anti-oxidant activities in alcoholic liver disease (ALD).
Aim of the study
P2X7 receptor (P2X7R) plays an important role in health and disease, which can be activated by extracellular ATP to induce a variety of downstream events, including lipid metabolism, inflammatory molecule release, oxidative stress. However, whether the mechanism of quercetin on ethanol-induced hepatic steatosis via P2X7R-mediated haven't been elucidated.
Material and methods
Zebrafish transgenic (fabp10: EGFP) larvae were treated with 100 μM, 50 μM, 25 μM quercetin for 48 h at 3 days post fertilization (dpf), then soaked in 350 mmol/L ethanol for 32 h, treated with 1 mM ATP (P2X7R activator) for 30min. Serum lipids, liver steatosis, oxidative stress factors were respectively detected. The mRNA levels in the related pathways were measured by quantitative Real-Time PCR (RT-qPCR) to investigate the mechanisms.
Results
Quercetin improved the liver function via decreasing ALT, AST and γ-GT level of zebrafish with acute ethanol-induced hepatic steatosis and attenuated hepatic TG, TC accumulation. Additionally, quercetin significantly reduced the MDA content and suppressed the ethanol-induced reduction of hepatic oxidative stress biomarkers GSH, CAT and SOD and significantly down-regulated the expression of P2X7R, and up-regulated the expression of phosphatidylinositol 3-kinase (PI3K), Kelch like ECH associated protein1 (Keap1), Nuclear Factor E2 related factor 2 (Nrf2). Moreover, ATP stimulation activated P2X7R, which further mediated the mRNA expressions of PI3K, Keap1 and Nrf2.
Conclusion
Quercetin exhibited hepatoprotective capacity in zebrafish model, via regulating P2X7R-mediated PI3K/Keap1/Nrf2 oxidative stress signaling pathway.
... Recently, numerous studies have confirmed that zebrafish can be used as valid models to evaluate liver toxicity (Huo et al., 2019;Zhang et al., 2017b). The early embryonic stages of hepatogenesis are similar to that of the mammalian liver (Field et al., 2003), and the structure and main physiological processes of the zebrafish liver is generally the same as in mammals (Hinton and Couch, 1998). Zebrafish larvae are virtually transparent during early life stages, and the morphological changes of liver can be evaluated visually without the need for dissection (Hill et al., 2012). ...
Macrolide antibiotics (macrolides) are among the most commonly prescribed antibiotics worldwide and are used for a wide range of infections, but macrolides also expose people to the risk of adverse events include hepatotoxicity. Here, we report the liver toxicity of macrolides with different structures in zebrafish. The absorption, distribution, metabolism, excretion and toxicology (ADMET) parameters of macrolide compounds were predicted and contrasted by utilizing in silico analysis. Fluorescence imaging and Oil Red O stain assays showed all the tested macrolide drugs induced liver degeneration, changed liver size and liver steatosis in larval zebrafish. Through RNA-seq analysis, we found seven co-regulated differentially expressed genes (co-DEGs) associated with metabolism, apoptosis and immune system biological processes, and two co-regulated significant pathways including amino sugar and nucleotide sugar metabolism and apoptosis signaling pathway. We found that only fosab of seven co-DEGs was in the two co-regulated significant pathways. fosab encoded proto-oncogene c-Fos, which was closely associated with liver diseases. The whole-mount in situ hybridization showed high transcription of c-Fos induced by macrolide compounds mainly in the liver region of zebrafish larvae. Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) leakage assays revealed that macrolides exerts significant cytotoxic effects on L02 cells. qRT-PCR and western blot analysis demonstrated macrolides also promoted human c-Fos expression in L02 cells. The c-Fos overexpression significantly reduced cell viability by using CCK-8 assay. These data indicate that hepatotoxicity induced by macrolides may be correlated with c-Fos expression activated by these compounds. This study may provide a biomarker for the further investigations on the mechanism of hepatotoxicity induced by macrolide drugs with different structures, and extend our understanding for improving rational clinical application of macrolides.
... When using whole-body microarrays the most influential tissue for the response remains undefined. In case of EHMC, data from the whole body (microarray) mainly reflected the results obtained by qRT-PCR in the brain (and to a lesser extent those obtained in testes), but interestingly, not those of the liver, which is often analysed (Pesonen and Andersson, 1991;Hinton and Couch, 1998;Hinton, 2000). This demonstrates that transcriptional effects of chemicals should be analysed by a multiple organ approach, and not by one tissue alone. ...
... 8 At the molecular level, the molecular mechanism of zebrafish liver development is consistent with that of mammals, and its hepatocellular function and histopathological changes in a variety of liver diseases are also very similar to those of humans. 9 Therefore, in this study, the liver fluorescent transgenic zebrafish at 72-h post-fertilization (hpf) was used as a model for liver toxicity evaluation to investigate the hepatotoxicity and toxicity mechanisms of isopsoralen, which is expected to provide the experimental support for the subsequent whole animal experiments and elucidation of the hepatotoxicity mechanism of isopsoralen at a molecular level. ...
Isopsoralen is the main component of the Chinese medicine psoralen, which has antitumour activity and can be used for the treatment of osteoporosis. However, the mechanism behind its hepatotoxicity has not yet been elucidated. In this study, the hepatotoxicity of isopsoralen was investigated using zebrafish. Isopsoralen treatment groups of 25, 50 and 100 μM were established. The mortality, liver morphology changes, levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), liver histopathology and mRNA levels of liver injury–related genes in zebrafish larvae were measured. The results showed that isopsoralen resulted in the development of malformed zebrafish, dose-dependent increases in ALT and AST, decreased liver fluorescence and weakened fluorescence intensity. Histopathological examination showed that high-dose isopsoralen caused a large number of vacuolated structures in the larvae liver. The polymerase chain reaction results showed a significant decrease in the mRNA levels of genes related to antioxidant capacity (lfabp, gstp2 and sod1) and drug transport (mdr1, mrp1 and mrp2), indicating that isopsoralen significantly inhibited liver antioxidant capacity and drug efflux capacity in zebrafish larvae. Isopsoralen is hepatotoxic to zebrafish larvae via inhibition of drug transporter expression resulting in the accumulation of isopsoralen in the body and decreased antioxidant capacity, leading to liver injury.
... (23) Transmission electron microscopy (TEM) showed that the zebrafish bile canaliculus is formed on the apical side of hepatocytes and sealed by tight junctions between the intrahepatic biliary cells and hepatocytes (Fig. 1I,K). (15,24,25) It was enriched with actin microvilli, similar to mammals. Using a polyclonal antibody raised against mouse ABCB11, (26) we detected Abcb11 protein in zebrafish hepatocytes at 120 hours postfertilization (hpf) (Fig. 1E-H). ...
Bile salt export pump (BSEP) adenosine triphosphate-binding cassette B11 (ABCB11) is a liver-specific ABC transporter that mediates canalicular bile salt excretion from hepatocytes. Human mutations in ABCB11 cause progressive familial intrahepatic cholestasis type 2. Although over 150 ABCB11 variants have been reported, our understanding of their biological consequences is limited by the lack of an experimental model that recapitulates the patient phenotypes. We applied CRISPR/Cas9-based genome editing technology to knock out abcb11b, the ortholog of human ABCB11, in zebrafish and found that these mutants died prematurely. Histological and ultrastructural analyses showed that abcb11b mutant zebrafish exhibited hepatocyte injury similar to that seen in patients with progressive familial intrahepatic cholestasis type 2. Hepatocytes of mutant zebrafish failed to excrete the fluorescently tagged bile acid that is a substrate of human BSEP. Multidrug resistance protein 1, which is thought to play a compensatory role in Abcb11 knockout mice, was mislocalized to the hepatocyte cytoplasm in abcb11b mutant zebrafish and in a patient lacking BSEP protein due to nonsense mutations in ABCB11. We discovered that BSEP deficiency induced autophagy in both human and zebrafish hepatocytes. Treatment with rapamycin restored bile acid excretion, attenuated hepatocyte damage, and extended the life span of abcb11b mutant zebrafish, correlating with the recovery of canalicular multidrug resistance protein 1 localization.
Conclusions:
Collectively, these data suggest a model that rapamycin rescues BSEP-deficient phenotypes by prompting alternative transporters to excrete bile salts; multidrug resistance protein 1 is a candidate for such an alternative transporter. (Hepatology 2018;67:1531-1545).
... Therefore, larval zebrafish studies can be performed with single milligrams of compound in microtiter plates, essentially allowing high-content in vivo information to be gathered in an in vitro format. The early embryonic stages of hepatogenesis are similar to that of mice (Field, Ober, Roeser, & Stainier, 2003), and the structure and function of the zebrafish liver is generally the same as in mammals (Hinton & Couch, 1998). Zebrafish have been used as a universal preclinical model organism for drug-hepatotoxicity screening in vivo (Vliegenthart, Tucker, Del Pozo, & Dear, 2014;Mesens et al., 2015). ...
... The teleost liver is one of the most sensitive organs to show alteration in biochemistry, physiology and structure following exposure to various types of environmental pollutants (Hinton and Couch 1998). Since the liver is the central site of numerous vital functions (i.e. ...
Bioaccumulation and ultrastructural alterations in gill and liver tissues of 3-month old Asian sea bass, Lates calcarifer, in response to copper were studied by transmission electron microscopy (TEM). Fishes were exposed to two sublethal concentrations of copper (6.83 ppm and 13.66 ppm) for a period of 28 days. Accumulation of copper were higher in the liver followed by gills. The damaging effects of the histoarchitecture of gill tissues in coper-exposed sea bass were hypertrophy and hyperplasia of epithelial cells, severe oedema, telangiectasia and secondary lamellar fusion. In addition extensive vasodilation with stretching and necrosis of pillar cells were also noted. The liver showed hydropic swelling of hepatocytes with nuclear pyknosis and chromatin condensation. Blood congestion in sinusoids and accumulations of lipid droplets in the hepatocytes at higher concentrations were observed.
... In addition, in fish as in higher vertebrates, the kidney performs an important function related to electrolyte and water balance and the maintenance of a stable internal environment (CENGIZ, 2006). Thus, many studies showed that different toxicants accumulate mainly in metabolic organs such as the liver and kidney (KARADEDE et al., 2004; OLIVEIRA RIBEIRO et al., 2005; JABEEN et al., 2011; YANCHEVA et al., 2014a, b; DE JONGE et al., 2015; VASEEM & BANERJEE, 2013) which can lead to many histological alterations (HINTON & COUCH, 1998; CENGIZ, 2006; POLEKSIĆ et al., 2010; STENTIFORD et al., 2003). Levels of heavy metals such as lead, copper, cadmium, and zinc in marine fish have been extensively documented in the primary literature (e.g., ROMÉO et al., 1999; ZAUKE et al., 1999; JUREŠA & BLANUŠA, 2003). ...
Water contamination (heavy metals, pesticides, POPs, etc.) is a serious environmental issue which has been raising lots of attention in the last decades because it can destroy aquatic ecosystems and hence, reduce biodiversity. In the field of ecotoxicology it is of main interest to investigate what the effects of organic and inorganic toxicants on different biological organization (cell, tissue, organism, population) are. Thus, many authors use different test organisms and particularly, fish. In the current study we aimed to present collected data from the last years which describe why fish is an appropriate species in terms of ecotoxicological research.
... A variety of structural arrangements have been described for the liver and biliary tree of fish. In general, fish do not possess the lobular structure and zonally oriented portal tracts observed in mammalian livers (Eurell and Haensly, 1982;Hampton et al., 1989;Hinton and Couch, 1998;Robertson and Bradley, 1992;Schar et al., 1985). Instead, the hepatocytes are commonly arranged into tubular elements. ...
... The teleost liver is one of the most sensitive organs to show alteration in biochemistry, physiology and structure following exposure to various types of environmental pollutants (Hinton and Couch 1998). Since the liver is the central site of numerous vital functions (i.e. ...
Surface ultrastructure of the gill and liver of 3-month-old Asian sea bass, Lates calcarifer, after copper exposure, was investigated by scanning electron microscopy (SEM). Fish samples were exposed to copper concentrations of 6.83 and 13.66 ppm (sublethal) for 28 days with parallel untreated control. These structures showed structural modifications in both low and high concentrations of copper exposure. Oedema, hyperplasia, desquamation, necrosis, epithelial lifting, lamellar fusion, collapsed secondary lamellae, curling of secondary lamellae and aneurism in the secondary lamellae were observed in gill tissues exposed to copper. Hepatic lesions related to cloudy swelling of hepatocytes, congestion, vacuolar degeneration, dilation of sinusoids and nuclear hypertrophy were evident in the exposed sea bass liver tissue.
... The liver is recognised as one of the most sensitive organs to indicate alteration in biochemistry, physiology and structure following contaminant exposure (Hinton and Couch, 1998). It is the main detoxification organ in fish and tends to accumulate metals and organic contaminants in higher concentrations than other tissues or the surrounding environment (Heath, 1995). ...
... A clear difference of trout and mammalian IHICs existed with respect to the ratios of myleoid to lymphoid cells: while the mammalian liver contains almost equal percentages of Kupffer cells and lymphocytes (see above), the IHICs of trout liver contained only 5% myeloid cells (see Fig. 4). The low percentage of myeloid cells might be explained by the apparent lack of Kupffer cells: neither in histological studies of trout liver, cells displaying the morphological criteria of Kupffer cells were detected (Hampton et al., 1989;Hinton and Couch, 1998) nor were they found in the high speed pellet (600 Â g) of isolated liver cells (Blair et al., 1990(Blair et al., , 1995. There exists evidence for the presence of a reticuloendothelial system in fish (Dalmo et al., 1997), but this function may be executed by cell types other than Kupffer cells. ...
... Oval cell hyperplasia, as described in the present study, has been described previously in trout exposed to carcinogens (Hendricks et al., 1984), in hepatic regeneration induced by partial hepatectomy (Okihiro and Hinton, 2000) and in hepatocarcinogenesis induced in other teleosts (Couch and Courtney, 1987;Hinton and Couch, 1998). ...
In mammalian species, profibrogenic cells are activated to become myofibroblasts in response to liver damage. Few studies have examined hepatic myofibroblasts and their role in liver damage in teleosts. The aim of the present study was to investigate the involvement of myofibroblast-like cells in rainbow trout (Oncorhynchus mykiss) with hepatic damage induced by aflatoxin B1 (AFB1). Histopathological and immunohistochemical analyses characterized alterations in the liver stroma during the carcinogenic process. Anti-human α-smooth muscle actin (SMA) and anti-human desmin primary antibodies were used in immunohistochemistry. Only the anti-SMA reagent labelled cells in trout liver. In the livers of control fish, only smooth muscle in blood vessels and around bile ducts was labelled. In the livers from AFB1-treated fish, SMA-positive cells were present in the stroma surrounding neoplastic lesions and in areas of desmoplastic reaction. These observations indicate that in teleosts, as in mammals, the myofibroblast-like cell is involved in fibrosis associated with liver injury. Chronic liver injury induced in trout by aflatoxin may provide a useful model system for study of the evolution of such mechanisms.
... If A. anguilla is unable to degrade MC-LR or if MC-LR is metabolized at a slower rate than it is ingested, toxin will probably accumulate in some organs of the fish. Liver in teleosts is one of the most sensitive organs to show alterations in biochemistry, physiology and histopathology following exposure to various environmental pollutants (Hinton & Couch, 1998). Moreover, the liver plays a fundamental role in energy synthesis and vitellogenesis in fishes (Nagahama, 1983). ...
... This may be attributed to the unique structure of the biliary tree in fishes: where canaliculi are much shorter than in humans and merge into bile preductules, characterized by the presence of bile preductular epithelial cells (BPDECs). [80][81][82] Electron microscopy revealed that BPDECs appear normal after EtOH treatment (not shown). Therefore, bile flow may be normal since bile preductules, not canaliculi, are the main bile conduit in the fish liver. ...
Abstract Fatty liver disease in humans can progress from steatosis to hepatocellular injury, fibrosis, cirrhosis, and liver failure. We developed a series of straightforward assays to determine whether zebrafish larvae with either tunicamycin- or ethanol-induced steatosis develop hepatic dysfunction. We found altered expression of genes involved in acute phase response and hepatic function, and impaired hepatocyte secretion and disruption of canaliculi in both models, but glycogen deficiency in hepatocytes and dilation of hepatic vasculature occurred only in ethanol-treated larvae. Hepatic stellate cells (HSCs) become activated during liver injury and HSC numbers increased in both models. Whether the excess lipids in hepatocytes are a direct cause of hepatocyte dysfunction in fatty liver disease has not been defined. We prevented ethanol-induced steatosis by blocking activation of the sterol response element binding proteins (Srebps) using gonzo(mbtps1) mutants and scap morphants and found that hepatocyte dysfunction persisted even in the absence of lipid accumulation. This suggests that lipotoxicity is not the primary cause of hepatic injury in these models of fatty liver disease. This study provides a panel of parameters to assess liver disease that can be easily applied to zebrafish mutants, transgenics, and for drug screening in which liver function is an important consideration.
... Zebrafish complete primary liver morphogenesis by 48 hour post-fertilization (hpf) and liver is fully formed and functioning by 72 hpf. With the exception of minor differences, the general anatomy, organization, cellular composition and function of a healthy zebrafish liver is virtually the same as in mammals (Hinton & Couch, 1998), and the early embryonic stages of hepatogenesis are similar to that of mice (Duncan, 2003;Field, Ober, Roeser, & Stainier, 2003;. Likewise, with regard to disease phenotypes, the histopathology of cholestasis, fatty liver (steatosis) and neoplasia is also comparable (Amali et al., 2006;Amatruda, Shepard, Stern, & Zon, 2002;Spitsbergen et al., 2000). ...
Introduction:
Numerous studies have confirmed that zebrafish and mammalian toxicity profiles are strikingly similar and the transparency of larval zebrafish permits direct in vivo assessment of drug toxicity including hepatotoxicity in zebrafish.
Methods:
Hepatotoxicity of 6 known mammalian hepatotoxic drugs (acetaminophen [APAP], aspirin, tetracycline HCl, sodium valproate, cyclophosphamide and erythromycin) and 2 non-hepatotoxic compounds (sucrose and biotin) were quantitatively assessed in larval zebrafish using three specific phenotypic endpoints of hepatotoxicity: liver degeneration, changes in liver size and yolk sac retention. Zebrafish liver degeneration was originally screened visually, quantified using an image-based morphometric analysis and confirmed by histopathology.
Results:
All the tested mammalian hepatotoxic drugs induced liver degeneration, reduced liver size and delayed yolk sac absorption in larval zebrafish, whereas the non-hepatotoxic compounds did not have observable adverse effect on zebrafish liver. The overall prediction success rate for hepatotoxic drugs and non-hepatotoxic compounds in zebrafish was 100% (8/8) as compared with mammalian results, suggesting that hepatotoxic drugs in mammals also caused similar hepatotoxicity in zebrafish.
Discussion:
Larval zebrafish phenotypic assay is a highly predictive animal model for rapidly in vivo assessment of compound hepatotoxicity. This convenient, reproducible animal model saves time and money for drug discovery and can serve as an intermediate step between cell-based evaluation and conventional animal testing of hepatotoxicity.
... The architectural pattern for mammalian liver is the one that is by far the most well-known and, while overall similarity exists in livers of various vertebrates, the mammalian pattern has been incorrectly applied to certain descriptions of hepatic structure of fish. Hinton and Couch (1998) stated that if we are to use aquatic species in toxicity testing it is imperative that we understand how tubular liver of teleosts responds to toxicants and there are at least six points of digression: 1) Livers of many fish are a single lobe enabling sampling of the entire organ thereby avoiding problems inherent with distribution and size of focal lesions common to two-dimensional studies. ...
... Some brown trout Salmo trutta f. fario L. contain cells (pale-grey cells) sharing structural and positional characteristics with the macrophages residing amidst hepatocytes (Rocha et al., 1994) and known as interhepatocytic macrophages (Hinton & Couch, 1998;Rocha & Monteiro, 1999). Despite these similarities, these two cell types are sufficiently different to question the positioning of pale-grey cells within the macrophage lineage (Rocha et al., 1996). ...
Pale-grey cells appeared in the livers of healthy and non- stressed brown trout Salmo trutta 3 weeks post-hatching whereas interhepatocytic macrophages appeared at 5 months. Cells with intermediate morphological characteristics between those of pale -grey cells and macrophages were identified in all ages studies. Cell ultrastructure supported the idea that pale-grey cells probably belonged to the macrophage lineage, being eventual precursors of the active phagocytes residing amongst hepatocytes. © 2002 The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved.
... Bile secreted by hepatocytes enters the centrally located ducts through canaliculus, which then join large ducts that carry bile to the extrahepatic biliary system and the gallbladder. Portal vein and hepatic vein branches are indistinguishable from one another (Hinton and Couch, 1998). As a matter of fact, the latter model is widely believed to be a common architecture existing in all embryonic vertebrates. ...
The fine structures, enzyme histochemical, and immunohistochemical characterization of liver in zebrafish were investigated using light microscopy and electron microscopy. The results showed that liver was separated into three lobes and each lobe had a central vessel comparable to the mammalian central vein. However typical hepatic lobules, portal areas, and hepatic arteries were not observed. A portal vein entered the liver and its tributaries were connected directly to the sinusoids, which then converged to the central vessel. Three central vessels in lobes finally carried the blood out of liver. The polygonal and bilayered hepatocytes were arranged as twisting, branching, and anastomosing cords. Ultrastructurally, they showed apparent morphological features of protein synthesis and secretion. Bile entered the biliary tree through the intracellular canaliculus, the ramifications of intercellular canaliculi that originated near the hepatic nucleus and then extended to the hepatocyte surface where two adjacent hepatocyte membranes formed intercellular canaliculi, and then ran sequentially through bile preductules, bile ductules, and bile ducts to be secreted out of the liver. Bile preductular epithelial cells (BPs) were cells located between bilayered hepatocytes in one hepatic cord. Occasionally, some tight junctions were detected forming the link between BPs and hepatocytes, which led us to assume that BPs might have a close relationship with hepatocytes during evolution. The present results indicate that zebrafish liver has its own specific fine structure.
... Development of a physiologically functional liver is therefore very rapid, in comparison to other vertebrate models. In addition, the structure and function of a healthy adult zebrafish liver is generally the same as in mammals (Hinton and Couch, 1998). For instance, bile is produced in the liver and, in the adult zebrafish, is stored in a gallbladder (Pack et al., 1996). ...
Drug-induced liver injury (DILI) is a major cause of attrition during both the early and later stages of the drug development and marketing process. Reducing or eliminating drug-induced severe liver injury, especially those that lead to liver transplants or death, would be tremendously beneficial for patients. Therefore, developing new pharmaceuticals that have the highest margins and attributes of hepatic safety would be a great accomplishment. Given the current low productivity of pharmaceutical companies and the high costs of bringing new medicines to market, any early screening assay(s) to identify and eliminate pharmaceuticals with the potential to cause severe liver injury in humans would be of economic value as well. The present review discusses the background, proof-of-concept, and validation studies associated with high-content screening (HCS) by two major pharmaceutical companies (Pfizer Inc and Jansen Pharmaceutical Companies of Johnson & Johnson) for detecting compounds with the potential to cause human DILI. These HCS assays use fluorescent-based markers of cell injury in either human hepatocytes or HepG2 cells. In collaboration with Evotec, an independent contract lab, these two companies also independently evaluated larval zebrafish as an early-stage in vivo screen for hepatotoxicity in independently conducted, blinded assessments. Details about this model species, the need for bioanalysis, and, specifically, the outcome of the phenotypic-based zebrafish screens are presented. Comparing outcomes in zebrafish against both HCS assays suggests an enhanced detection for hepatotoxicants of most DILI concern when used in combination with each other, based on the U.S. Food and Drug Administration DILI classification list.
... There was no appreciable difference in the appearance of liver sections in control and pk1a MO-injected larvae by hematoxylin and eosin staining (data not shown). Given the lack of a lobular structure and portal triads in teleost larvae and adults (Hinton and Couch, 1998), differences may be more difficult to appreciate by this method, however. Thus, we examined larvae by electron microscopy. ...
Planar cell polarity (PCP) establishes polarity within an epithelial sheet. Defects in PCP are associated with developmental defects involving directional cell growth, including defects in kidney tubule elongation that lead to formation of kidney cysts. Given the strong association between kidney cyst formation and developmental biliary defects in patients and in animal models, we investigated the importance of PCP in biliary development. Here we report that in zebrafish, morpholino antisense oligonucleotide-mediated knockdown of PCP genes including prickle-1a (pk1a) led to developmental biliary abnormalities, as well as localization defects of the liver and other digestive organs. The defects in biliary development appear to be mediated via downstream PCP targets such as Rho kinase, Jun kinase (JNK), and both actin and microtubule components of the cytoskeleton. Knockdown of pk1a led to decreased expression of vhnf1, a homeodomain gene previously shown to be involved in biliary development and in kidney cyst formation; forced expression of vhnf1 mRNA led to rescue of the pk1a morphant phenotype. Our results demonstrate that PCP plays an important role in vertebrate biliary development, interacting with other factors known to be involved in biliary morphogenesis.
... In this way, liver developed a peculiar morphology, with interrelating stromal and parenchymal components made of different cell types, all strategically positioned and organized. Since the middle of the late century, fish hepatic histology and cytology have been the subject of numerous works (Elias and Bengelsdorf, 1952;Hampton et al., 1985;Beresford and Henninger, 1986;Hinton and Couch, 1998) and a crescent focus of interest. ...
Doutoramento em Ciências Biomédicas
... Liver in Teleost fishes is one of the most sensitive organs to show alteration in biochemistry, physiology and structure following exposure to various types of environmental pollution (Hinton & Couch, 1998). Based on this, silver eels (i.e. ...
Population dynamics of European eels Anguilla anguilla was investigated within a small dammed catchment of Britanny (the Fremur) over a 8-years period (from 1996 to 2003). On this period, the migrant (recruitment and escapement) and sedentary (stock) fractions were quantified and characterized (stage, length and age). The mortality rate observed was important (53.2%) probably because of the high abundance (0.40 eels.m-2) and the habitatloss. Each year, the number of potentially emigrant eels, based on an analysis of the sedentary population fraction, was predicted. The observed number was strongly dependent on environmental conditions that were overruled by dam management. Finally, influence of growth site on production of both number and quality of potential breeders, and implications of this study for European eel conservation, were discussed. L'anguille européenne Anguilla anguilla (L.) occupe des habitats dulçaquicoles, saumâtres et côtiers dans les tous les pays de l'Europe et le long des côtes méditerranéennes. Son cycle de vie n'est pas complètement connu mais les preuves disponibles indiquent que les anguilles colonisant les eaux continentales sont issues d'un stock unique de géniteurs de l'Océan Atlantique. Ce stock est considéré maintenant comme en dehors de ses limites de sécurité biologique. Les causes réelles du déclin sont inconnues mais des hypothèses continentales et océaniques, d'origine naturelles et anthropiques ont été suggérées. En phase continentale où des actions de gestion sont possibles, le stock est fragmenté en une multitude de sous populations indépendantes. Cependant, les évaluations du nombre et de la "qualité" des futurs reproducteurs produits dans ces hydrosystèmes, font défaut. Dans cette étude, nous examinons sur une période 8 années la dynamique continentale d'une sous population d'un petit fleuve côtier (60 km²) fortement aménagé du Nord de la Bretagne, le Frémur. Sur cette période, les trois écophases clés (Recrutement, Stock et Dévalaison) ont été quantifiées en relation avec les facteurs environnementaux et caractérisées (taille, age, sexe et stades). Le taux instantané de disparition (mortalité + émigration) a été évalué à 0,761 ce qui correspond à une diminution annuelle de 53,2% des effectifs entre 3 et 6 ans. La capacité d'accueil du bassin versant du Frémur, probablement atteinte, en raison d'abondances d'anguilles très importantes (0,40 anguilles.m-2) et des habitats dégradés, peut expliquer ce résultat. De plus, nous montrons qu'il est possible de prédire chaque année le nombre potentiel de futurs reproducteurs à entreprendre leur migration (Dévalaison) à partir de la connaissance de la structure (taille, abondance et stade) des anguilles sédentaires (Stock). Le nombre effectif variant en fonction des conditions environnementales et surtout de la gestion hydraulique du système. Enfin, une comparaison de la condition moyenne des anguilles migrantes produites dans le Frémur avec des individus issus d'un bassin versant de taille équivalente mais non anthropisé, révèle une qualité inférieure des anguilles du Frémur. Finalement, l'influence du contexte de croissance (productivité, qualité de l'eau, barrages, etc.) sur la production du nombre et de la qualité des futurs reproducteurs, et les implications de cette étude en matière de conservation de cette espèce à l'échelle européenne, sont discutées. Ce programme de recherche a bénéficié du financement : de la Fédération des AAPPMA d'Ille et Vilaine (Maître d'Ouvrage) du Conseil Régional de Bretagne du Ministère de l'Aménagement du Territoire et de l'Environnement de l'Agence de l'Eau Loire Bretagne du Conseil Général d'Ille et Vilaine du Conseil Général des Côtes d'Armor.
... In addition, it has been proposed that the possibility of detecting Vtg in the surface mucus could result in a useful tool for monitoring the presence of estrogenic pollutants in the environment (Moncaut et al., 2003;Meucci and Arukwe, 2005;Van Veld et al., 2005;Maltais and Roy, 2007;Arukwe and Røe, 2008). On the other hand, the teleost liver is one of the most sensitive organs to show alterations in biochemistry, physiology and structure following exposure to various types of environmental pollutants (Hinton and Couch, 1998;Andersson et al., 2007;Perez Carrera et al., 2007). ...
Exposure to environmental pollutants may disrupt endocrine functions and cause reproductive effects in human and wildlife populations. Various groups of chemicals have estrogen-like effects, including degradation products of alkylphenol polyethoxylates, such as 4-tert-octylphenol (OP). Laboratory studies have shown that exposure of male fish to xenoestrogens results in induction of circulating vitellogenin (Vtg), inhibition of testicular growth, testis abnormalities and formation of intersex gonads. In this study, the impact of the exposure to waterborne OP on reproductive aspects in the South American cichlid fish Cichlasoma dimerus was evaluated using qualitative changes in the levels of Vtg in plasma and surface mucus and histological alterations in the liver and gonads as endpoints. Adult males and females were exposed to OP via immersion during 60 days in aquaria under semi-static conditions, water changes being made every 84 h. Treatment groups were: control (ethanol 0.005%), OP 30, 150 and 300 microg/L. Using Western and Dot blot analysis, Vtg was detected in plasma and mucus of control and treated females and treated males, while no Vtg was observed in samples from control males. Morphological changes in the hepatocytes due to the accumulation of Vtg were observed in OP-exposed males. Impairment of testicular structure became apparent in males treated with the highest OP concentrations. The most salient pathological change was the alteration of lobular organization with increased testicular fibrosis and progressive disruption of spermatogenesis. No major changes were observed in ovarian architecture. Our results indicate that detection of Vtg in surface mucus may be a sensitive and non-invasive biomarker of the endocrine disrupting effects of environmental estrogens, resulting in a useful method for field monitoring.
... The majority of our understanding of vertebrate hepatobiliary disease and toxicity has been derived from mammalian liver studies [1][2][3][4][5]. We know comparatively less about piscine biliary disease and toxicity, though we are beginning to gain greater insight into piscine hepatobiliary structure/function relationships [6][7][8][9][10][11][12][13][14][15][16][17]. Because our understanding of the piscine biliary system has lagged, particularly in a comparative sense, our ability to interpret and communicate biliary disease and toxicity in aquatic species has remained limited. ...
A novel transparent stock of medaka (Oryzias latipes; STII), recessive for all pigments found in chromatophores, permits transcutaneous imaging of internal organs and tissues in living individuals. Findings presented describe the development of methodologies for non invasive in vivo investigation in STII medaka, and the successful application of these methodologies to in vivo study of hepatobiliary structure, function, and xenobiotic response, in both 2 and 3 dimensions.
Using brightfield, and widefield and confocal fluorescence microscopy, coupled with the in vivo application of fluorescent probes, structural and functional features of the hepatobiliary system, and xenobiotic induced toxicity, were imaged at the cellular level, with high resolution (< 1 microm), in living individuals. The findings presented demonstrate; (1) phenotypic response to xenobiotic exposure can be investigated/imaged in vivo with high resolution (< 1 microm), (2) hepatobiliary transport of solutes from blood to bile can be qualitatively and quantitatively studied/imaged in vivo, (3) hepatobiliary architecture in this lower vertebrate liver can be studied in 3 dimensions, and (4) non invasive in vivo imaging/description of hepatobiliary development in this model can be investigated.
The non-invasive in vivo methodologies described are a unique means by which to investigate biological structure, function and xenobiotic response with high resolution in STII medaka. In vivo methodologies also provide the future opportunity to integrate molecular mechanisms (e.g., genomic, proteomic) of disease and toxicity with phenotypic changes at the cellular and system levels of biological organization. While our focus has been the hepatobiliary system, other organ systems are equally amenable to in vivo study, and we consider the potential for discovery, within the context of in vivo investigation in STII medaka, as significant.
... Whereas medaka have been shown to be sensitive subjects for carcinogenesis research, several differences exist between fish and mammals with respect to normal liver histomorphology (11). In addition to the absence of lobes, fish livers do not have portal triads and as such have no histologically defined acini or lobules. ...
Progression of hepatic neoplasia was assessed in medaka (Oryzias latipes) following aqueous exposure to diethylnitrosamine (DEN). Larvae (2 weeks old) were exposed to 350 or 500 p.p.m. DEN for 48 h, while adults (3-6 months old) were exposed to 50 p.p.m. DEN for 5 weeks. Fish were maintained as long as possible to determine malignant potential of resultant neoplasms. A total of 423 medaka with 106 hepatic neoplasms were examined. There were marked differences in tumor prevalence between exposure groups including: (i) higher prevalence of hepatocellular carcinomas in medaka exposed as adults (100% of hepatocellular tumors in adult-exposed medaka were malignant, while only 51.5% of larval hepatocellular tumors were malignant); (ii) higher prevalence of biliary tumors in medaka exposed as larvae (46.4% of all tumors in larval-exposed medaka were biliary versus 8.1% in adult-exposed fish); (iii) higher prevalence of mixed hepato-biliary carcinomas in adult-exposed medaka (24.3%) compared with those exposed as larvae (3%). In addition, a unique hepatocellular lesion termed 'nodular proliferation' was only observed in adult-exposed medaka. The lesion was characterized by small size (50-300 microm), complete loss of normal tubular architecture and variable megalocytosis. Nodular proliferation was distinct from preneoplastic foci of cellular alteration and may represent microcarcinomas. There was a step-wise increase in mean diameter with age (days post-exposure) from nodular proliferation (174 microm, 17 days) to hepatocellular carcinoma (1856 microm, 62 days) and mixed carcinomas (3209 microm, 93 days) in adult-exposed medaka. Metastasis was observed with 19 neoplasms and tumors with the highest metastatic potential were hepatocellular and mixed carcinomas. The most common form of metastasis was trans-coelomic, followed by direct invasion and distant metastasis, presumably via the vascular route. Differences in tumor prevalence between exposure groups were believed to be the result of length of DEN exposure rather than age of fish at the time of exposure. In larval medaka with brief (48 h) DEN exposure, neoplasms are thought to be the result of dedifferentiation of hepatic cells, with slow progression of foci of cellular alteration to benign and then malignant tumors. In contrast, with adult medaka and prolonged (5 week) DEN exposure, neoplasms are believed to result from initiation of committed stem cells and formation of microcarcinomas ('nodular proliferation'), before progressing to larger hepatocellular and then mixed carcinomas.
... Livers of fishes are similar to those of mammals, but there are important differences (23)(24)(25). A fundamental difference is the glandular arrangement of cells within the teleost liver, which translates into an abundance of biliary epithelial cells in close proximity to hepatocytes within the parenchymal compartment (27,28). Whereas hepatocytes of mammalian liver are arranged as a continuum of laminae (2), analogous structures of teleost livers are tubules of hepatocytes and biliary epithelial cells, with lumens and mural elements of hepatocellular tubules providing the architectural framework for an intrahepatic biliary tree. ...
Hepatic regeneration following partial hepatectomy (PH) and biliary hyperplasia subsequent to bile duct ligation (BDL) were characterized in rainbow trout (Oncorhynchus mykiss) by light microscopy using routine and special (immunohistochemical and enzyme histochemical) stains. Both PH and BDL involved initial hypertrophy and hyperplasia of bile preductular epithelial cells (BPDECs). BPDECs are small oval cells that form junctional complexes with hepatocytes and bile ductular cells and are commonly found in hepatic tubules of teleost liver. Proliferating BPDECs transitioned through intermediate cell types before final differentiation into large basophilic hepatocytes (following PH) or biliary epithelial cells (after BDL). Normal BPDECs and hepatocytes were both negative for cytokeratin intermediate filaments in control fish when screened with the monoclonal antibody AE1/AE3. In contrast, hyperplastic BPDECs and their progeny (intermediate cells, immature hepatocytes, ductal epithelial cells) were all strongly cytokeratin positive. Cytokeratin expression was transient in newly differentiated hepatocytes (expression decreased as hepatocytes acquired characteristics consistent with full differentiation) but was permanent in biliary epithelial cells (expression was very strong in large mature ducts). BPDECs, intermediate cells, and immature ductal cells were also strongly positive for alkaline phosphatase following BDL. Chronology of histologic events and cytokeratin and enzyme expression all support the hypothesis that BPDECs possess the capacity to differentiate into either hepatocytes or biliary epithelial cells. Thus, BPDECs may be the teleost equivalent of a bipolar hepatic stem cell in mammals.
Zebrafish have become a powerful model of mammalian lipoprotein metabolism and lipid cell biology. Most key proteins involved in lipid metabolism, including cholesteryl ester transfer protein, are conserved in zebrafish. Consequently, zebrafish exhibit a human-like lipoprotein profile. Zebrafish with mutations in genes linked to human metabolic diseases often mimic the human phenotype. Zebrafish larvae develop rapidly and externally around the maternally deposited yolk. Recent work revealed that any disturbance of lipoprotein formation leads to the accumulation of cytoplasmic lipid droplets and an opaque yolk, providing a visible phenotype to investigate disturbances of the lipoprotein pathway, already leading to discoveries in microsomal triglyceride transfer protein and ApoB (apolipoprotein B). By 5 days of development, the digestive system is functional, making it possible to study fluorescently labeled lipid uptake in the transparent larvae. These and other approaches enabled the first in vivo description of the STAB (stabilin) receptors, showing lipoprotein uptake in endothelial cells. Various zebrafish models have been developed to mimic human diseases by mutating genes known to influence lipoproteins (eg, ldlra , apoC2 ). This review aims to discuss the most recent research in the zebrafish ApoB-containing lipoprotein and lipid metabolism field. We also summarize new insights into lipid processing within the yolk cell and how changes in lipid flux alter yolk opacity. This curious new finding, coupled with the development of several techniques, can be deployed to identify new players in lipoprotein research directly relevant to human disease.
The demand to predict the toxicity before the clinical trials increased due to the continuous failure of several products after marketing. This led to a preclinical animal study that mimics the human body more and produces results rapidly and accurately. The zebrafish model was one such model that gave extensive, similar results to that of human conditions. Due to the number of advantages, this model became an ideal alternative for various studies where a large animal model cannot be preferred. Also, this model has been recently identified as the unique model in investigating several narrow therapeutic drugs and highly toxic drugs, proving its capability to determine several characteristics addressed in cancer and neurodegenerative disorders.
Histopathology can reveal toxicant‐induced changes in the structure of a tissue or organ. A prerequisite for histopathological studies is a sound knowledge of the morphology of the anatomical structure in the normal or healthy state. Zebrafish larvae can provide a tool for studies focused on hepatotoxicity at early stages of development; therefore, the fine structure of the organ should be well characterised. To date, liver structure at 72 and 120 hr post‐fertilisation (hpf) has not been reported in detail and this study aimed to fill this scientific gap. A stereological approach allowed for quantitative description of the liver and revealed ultrastructural alterations occurring with time of development. These included a significant increase in the absolute volume of hepatocytes, mitochondria and rough endoplasmic reticulum (rER) during the period of study. The surface area of rER, and of outer and inner mitochondrial membranes also increased. There was no change in the absolute volume of the nuclei. This study provides a quantitative spatial and temporal framework for future research aiming to detect early developmental changes in the liver. Zebrafish larvae can provide a tool for studies focused on hepatotoxicity at early stages of development. In the present study hepatocyte ultrastructure was quantified using a stereological approach at 72 and 120 hr post‐fertilization. This study provides a quantitative spatial and temporal framework for future research aiming to detect early developmental changes in the liver.
Zebrafish: Methods for Assessing Drug Safety and Toxicity offers a practical guide for using zebrafish as a tool for toxicology studies. Consolidating key protocols and approaches to help researchers navigate the important and evolving field of zebrafish models for toxicity screening, this new title describes the methods for using the zebrafish as a model organism to assess compound-induced toxicity on all major organs. Individual chapters that concentrate on assays for each organ system are included and various analytical tools including microscopy, microplate readers, high content imaging systems, ECG, blood pressure monitors, high speed video and motion detectors are described.
Cadmium (Cd) is one of the most harmful heavy metal pollutants in aquatic environments. Fingerlings of Nile tilapia, Oreochromis niloticus, were exposed to sublethal concentrations (10, 20 and 30% of the 96-h LC 50) CdCl 2 which caused significant changes in concentration dependent manner in the hepatic and ileal tissues. The hepatic tissue lost its characteristic architecture, with increased vacoulations in hepatocytes. In addition, abundant eythrocytic infiltration was observed in the 10% of LC 50 group and clearly increased in the 20% of LC 50 group. Increase of hemorrhage, vacuolation in hepatocytes and infiltration of sinusoids with leukocytes were observed in the 30% of LC 50 group. The intestinal tissue in the treated groups was characterized by increased degenerated nuclei and apoptosis in crypts of Lieberkuhn, in addition to abnormally dilated lamina propria infiltrated with a large number of inflammatory leukocytes and disturbance of the longitudinal muscularis. Goblet cells increased in all treated groups, indicating a defense mechanism against the severe pathological changes induced. In conclusion, severe damage to the hepatic and intestinal tissues of the Nile tilapia was observed upon contamination of the fish environment with CdCl 2 .
The fish liver has been the main subject of the biomonitoring and laboratory studies dealing with environmental carcinogenesis. The foci of cellular alterations are accepted pre-neoplastic hepatic lesions, and histopathology is the primary tool for their characterization. Despite its potential, using stereology to study quantitatively nuclear features of those lesions has not been evaluated. Herein, we estimated the volume density and the volume-weighted volume of the nucleus of normal and preneoplastic hepatocytes, using stereology and the brown trout (Salmo trutta f. fario) as model. In the hepatocarcinogenesis protocol the N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was used as initiator, and the 17-β estradiol (E2) as promoter. Three groups of 30 animals were considered: negative controls (non-exposed), initiator exposed and initiator plus promoter exposed. Estimates of both stereological parameters were significantly higher in preneoplastic hepatocytes, also showing an excellent discriminatory power when used to differentiate those hepatocytes from the normal ones. Besides, in the normal parenchyma the two parameters also differed among the three tested groups. The exposure to MNNG and/or to E2 leaded to modifications in the hepatocyte nuclei that could be unbiasedly quantified with two stereological parameters. We showed that quantitative nuclear morphology represents a valuable auxiliary tool in assessing hepatocarcinogenesis in fishes.
Thirty migrating silver eels Anguillaanguilla were collected in a river system where algal blooms occurred yearly. Fifty per cent of eel livers were contaminated by microcystin-LR (mean ±s.d. toxin level: 28·1 ± 22·4 ng g−1). Contaminated silver (v. healthy) eels had lower fish condition. Consequences of this impact for the breeding potential of these migrating eels are discussed.
The study investigates the influence of different culture conditions on attachment, viability and functional status of rainbow trout (Oncorhynchus mykiss) liver cells in primary culture. Cells were isolated by a two-step collagenase perfusion and incubated in serum-free, chemically defined minimal essential medium (MEM), (a) as a monolayer on uncoated PRIMARIA dishes, (b) as a monolayer on culture dishes coated with calf collagen type 1, and (c) in coculture with the established fish cell lines RTH-149 or RTG-2. Cell attachment was assessed from DNA and protein concentrations per dish, viability was estimated from cellular lactate dehydrogenase release, and the metabolic status was investigated by measuring activities of the phosphoenolpyruvate carboxykinase and biotransformation enzymes as well as the total cytochrome P450 contents. Seeding of hepatocytes on collagen-coated dishes did not alter cell attachment or detachment from the (culture substrate, but had a small, but not significant effect on cell viability and metabolic parameters. Coculture of liver cells and RTG-2 cells reduced hepatocyte detachment from the culture substrate, and it was associated with a significant elevation of 7-ethoxyresorufin-O-deethylase activities in the hepatic cells. Cytochrome P450 contents, however, were not altered. The coculture effect on liver cell physiology clearly depended on the type of cell line, because coculture with RTH-149 cells led to similar, but much weaker effects than obtained in cocultures with RTG-2 cells. Electron microscopical observations revealed the existence of gap junctions and possible exocytosis-like transport between cell lines and hepatocytes. The results point to the potential of coculture systems to improve physiological parameters of trout liver cells in primary culture.
Liver transplantation has been regarded as the definitive curative approach for pathologic liver conditions from the acute stage to the chronic end stage for decades. Recently, translational research has been focused on liver stem cell transplantation, using various cell therapies, due to the potential benefit of natural host liver regeneration. Many studies are ongoing utilizing and evaluating the use of either fetal-liver-derived stem cells or oval cells, however many obstacles still remain. Extensive research identifying and characterizimg stem/progenitor cells for potential application to in vitro cell therapy, whereas many questions remain concerning the isolation and identification of adult liver stem cells with adequate capacity for proliferation and the regeneration of injured liver. Recent approaches to liver regeneration include the production of hepatocyte-like cells from other stem cell sources such as mesenchymal stem cells and embryonic stems cells. Another major target for liver regeneration studies include the generation of liver stem cells from induced pluripotent stem cells (IPSC) We review the current data concerning characterization of stem cells and progenitor cells for their capacity to support their potential for re-population and regeneration of normal adult liver from liver damaged due to injury and/or disease.
A positive immunocytochemical reaction for the N-terminal peptide of N-ras oncoprotein was detected in small groups of hepatocytes and circular foci of altered cells in all the livers of dab (Limanda limanda) sampled from a contaminated site in the German Bight. Fish sampled from less contaminated sites showed a low or zero incidence of N-ras positive epitope. Livers of fish from the contaminated site were known to have a high incidence of putative preneoplastic lesions and the possible use of ras oncoprotein as a tumour marker in environmental toxicologic pathology is discussed.
Adult Oryzias latipes were exposed to 50 mg of diethylnitrosamine per liter of water for 5 wk and then transferred to clean water for an additional 15 wk. Response of the liver during the first 6 wk were analyzed by enzyme histochemistry and by high-resolution light and transmission electron microscopy. After 1 wk, cytotoxicity was apparent at the light microscopic level by piecemeal necrosis and phagocytosis apoptosis by adjacent hepatocytes and resident macrophages. Spongiosis hepatis and inflammation, found as early as wk 3, were not widespread until wk 6. Glycogen depletion and multifocal increases in gamma-glutamyl transpeptidase were found as early as 3 wk. At 5 wk, macrophage infiltration and aggregation and hepatocyte lysosome proliferation were revealed by an increase in cells staining for acid phosphatase. In addition, a subpopulation of macrophages stained positively for glucose-6-phosphate dehydrogenase during wk 6. Other histochemical biomarkers (Mg2(+)-ATPase, DT-diaphorase, uridine diphosphoglucuronyl dehydrogenase) were not altered. Mitotic figures were rare for the entire 6-wk period. At the ultrastructural level, necrotic alterations of some hepatocytes were seen within 24 h. Within 48 h, an apparent reduction of hepatocyte glycogen and cell volume characterized the majority of hepatocytes; this was accompanied by an increase in interhepatocytic space and the length and complexity of the hepatocyte microvillous projections found in the space of Disse. Lipid vacuolar inclusions inhabited space previously occupied by glycogen. Margins of hepatocyte nuclei were irregular, and mitochondria were condensed and their shape altered so that crescentric and elongated profiles were abundant. Lysosomes and residual bodies were increased after 1 wk. The cytoplasmic processes delineating spongiotic lesions were identified as originating from Ito cells. After 4 wk, apparent proliferation of smooth endoplasmic reticulum and retention of transport lipid within its cisternae were seen. The toxic depletion of hepatocytes and the attendant altered cellular environment are discussed in relation to cell-to-cell interactions and the possible contribution of stromal and extracellular matrix changes to liver regeneration and neoplasia.
Critical parameters in the quantitation of altered hepatic foci (AHF) developing during multistage hepatocarcinogenesis in the rat include: 1) the enumeration of AHF induced by test agents as well as those AHF occurring spontaneously in livers of untreated animals; 2) the volume percentage or fraction of the liver occupied by all AHF as a reflection of the total number of altered cells within the liver and the degree of tumor promotion which has occurred; and 3) the phenotype of individual AHF as determined by multiple markers with serial sections. These parameters, especially the number of AHF, should be corrected by the presence of spontaneous AHF which increase with the age of the animal, more so in males than females. While accurate estimation of the background level of spontaneous AHF can be important in demonstrating that a carcinogenic agent does not possess the ability to increase the numbers of AHF above the background level, a better method to distinguish the effectiveness and relative potencies of agents as initiators or promoters is reviewed. The relative effectiveness of four different markers--gamma-glutamyltranspeptidase (GGT), a placental form of glutathione S-transferase (GST), canalicular ATPase, and glucose 6-phosphatase (G6Pase)--was described for the chemicals C.I. Solvent Yellow 14 and chlorendic acid as promoting agents in males and females. C.I. Solvent Yellow 14 is a more effective promoting agent in females than males, and AHF exhibit extremely low numbers scored by GGT. On the other hand, the numbers of AHF present in livers of male rats promoted by this agent are more than twice those seen in livers of female animals, possibly owing to the effectiveness of this agent as an initiator in the male but not the female. Very few AHF, especially in the male, are scored by GGT during chlorendic acid promotion. The distribution of phenotypes with these markers also differs in the spontaneous AHF appearing in the livers of animals fed 0.05% phenobarbital on either a crude NIH-07 or AIN-76 purified diet. Such studies emphasize the extreme dependence of the promoting stage of hepatocarcinogenesis on environmental factors of sex, diet, and the molecular nature of the promoting agent itself. The hallmark of the final stage of progression in the development of hepatocellular carcinomas is aneuploidy, which may be reflected by phenotypic heterogeneity within individual AHF, termed foci-in-foci. The implications of such quantitative analyses during hepatocarcinogenesis induced by specific agents in relation to the specific action of the agent at one or more of the stages of hepatocarcinogenesis are discussed.
When this book was first published in 1996, the pollution of the earth's freshwater habitats was, as it is now, a topic of major concern. This synthesis considers the effects of pollutants on aquatic animals via a series of research and review articles that present experimental evidence of sublethal and lethal effects of a range of toxicants at the physiological, cellular and subcellular levels, and that explore techniques for detection of pollution damage. Topics covered include routes of uptake of toxicants; the effect of acute and chronic exposure to toxic metal ions, particularly zinc, copper and aluminium, with emphasis on the mechanisms of toxicity and responses to chronic exposure to sublethal levels; the impact on fish biology of two chemicals of current concern, nitrites and polyaromatic hydrocarbons, which may act as oestrogenic substances or potent mutagens; and in vitro studies of the mechanisms of toxicity at the cellular and subcellular level, including damage of DNA, using cultured fish cells.
Seen by scanning electron microscopy (SEM), the adult mammalian liver appears to be a parenchymatous organ in which five principal types of fixed cells, each with specific morphofunctional features, are integrated to form a highly complex structure1,2. These cells may be classified as follows:
(1)
Epithelial parenchymal cells, or hepatocytes, arranged in one-cell-thick laminae dispersed between the portal space and the central vein;
(2)
Epithelial biliary cells making up the monolayered wall of the biliary ductules and biliary ducts;
(3)
Endothelial cells which, with their large and fenestrated cytoplasmic extensions, contribute toward the structural formation of the wall of the liver capillaries (the sinusoids);
(4)
Kupffer cells, which are the fixed macrophages within the liver sinusoids; and
(5)
Perisinusoidal cells (stellate, fat-storing or Ito cells), commonly found within the perisinusoidal space or space of Disse.
The mammalian liver is a large exocrine gland connected to the small intestine through the choledochus and to the splanchnic blood circulation by the portal vein and hepatic artery. It is located in the right upper quadrant of the abdomen, just beneath the diaphragm and extends to the hypogastrium and to the left hypochondrium. Topographically, it is divided into two larger (right and left) and two smaller (caudate and quadrate) lobes. Functionally, it can be divided in subsegments (not corresponding to the above-mentioned lobes) according to the intraparenchymal ramification of the blood vessels and biliary tree.
Foci of altered hepatocytes are not visible with the naked eye.
This chapter describes the different aspects of vitellogenesis and oocyte assembly. The major components of fish oocytes are derived from the blood-borne high-molecular-weight compound, vitellogenin, which is synthesized in the liver of oviparous vertebrates. The classification of vitellogenin as a phospholipoglycoprotein indicates the crucial functional groups that are carried on the protein backbone of the molecule—namely, lipids, some carbohydrates, and phosphate groups. In the rainbow trout, estrone administration leads to the induction of vitellogenin synthesis in the liver and its release into the bloodstream, but estrone displays only 5%–12% of the potency of estradiol. The biochemical information concerning vitellogenin clearly indicates that a great deal of posttranslational modification must occur in the liver cell to reach the finished product seen in the serum. An integral part of estradiol action is the observed hypercalcemia in vitellogenic fish, which can largely be ascribed to the calcium-binding properties of phosphorylated and highly charged, components of the native vitellogenin molecule.
The adaptive response of the omnivorous ide,Leuciscus idus melanotus, to drastic metabolic conditions was analyzed on different levels of organisation investigating a variety of parameters: Organism (condition factor, liver-somatic index), organ (liver structure), cellular and subcellular level (hepatocyte structure, glycogen and lipid storage, contents and distribution of nucleic acids, enzyme alterations).
During starvation, ide were able to maintain liver integrity in a biphasic process: after an initial phase of disturbance, ide established a new structural and metabolic homeostasis. Recovery from starvation was possible only with a complete diet but not with a sucrose diet. Carbohydrate overload, as evoked by sucrose refeeding, did not result in liver or carcass fattening as known from mammals.
To the best of our knowledge, the present study is the first to use enzyme histochemistry in fish nutrition research. In mammals, histochemistry is particularly helpful for understanding processes of hepatic metabolic adaptation. In fish, however, on the basis of our results, enzyme histochemical studies appear to be of limited value, as long as no further data are available on a zonal distribution of enzyme activities in teleost liver parenchyma. Instead, the histochemical detection of the distribution of hepatic storage products and RNA-positive material yielded important information on liver adaptive processes.
This paper reports the detailed histopathological analysis of livers of flounder Platichthys flesus sampled in Dutch coastal and estuarine waters during 1985-89. In conjunction with an epizootiological study of grossly identifiable diseases st 10 sampling locations, a total of 210 livers (1 % of sampled population) showing gross nodular lesions (diameter >2 mm) and 315 livers showing no gross pathology were collected. Of the 210 livers with grossly observable nodules, 67 % were diagnosed as having neoplasms. The majority of these neoplasms were identified as hepatocellular adenoma; 13.1 % were diagnosed as hepatocellular carcinoma. Most of the remaining nodules were diagnosed as foci of cellular alteration, which are considered to be preneoplastic lesions. Routine histopathological examination of livers showing no gross pathology resulted in the identification of a range of lesions including hepatocellular adenoma, foci of cellular alteration, hydropic vacuolization of biliary epithelial cells and hepatocytes, inflammatory lesions, focal necrosis, regenerative foci, and fibrillar hepatocytes of unknown significance. In addition, indices were used to quantify the presence of storage vacuoles (glycogen and lipid) and the relative density of melanomacrophage centres. The results indicate that a small proportion of neoplasms would be missed if only livers with grossly detectable nodules were subjected to histological examination. The prevalence of foci of cellular alteration at the different sampling sites showed a good correspondence with that of neoplasms, providing support for the hypothesis that these conditions represent stages of the same process. Of the other lesions and quantitative indices, only hydropic vacuolization of biliary epithelial cells had a spatial distribution similar to that of neoplasms. It is concluded that liver neoplasms and especially their precursor lesions in flounder are promising tools for monitoring exposure to potential carcinogens, provided that migration patterns are explicitly taken into account. Furthermore, experimental studies are needed to investigate the chemical agents responsible for the onset of these early lesions and their progression into neoplasms, and also to evaluate the contribution of other environmental and host-related factors.
In order to investigate potential links between marlne pollution and fish diseases, an ep~zootiological study was conducted in The Netherlands during 1983-89. This study concentrated on grossly identifiable diseases of flounder Platichthys flesus. Flounder were found to be affected by the viral skin disease lymphocystis and by skin ulcers probably of bacterial origin. Overall prevalences of these 2 diseases in fish 2 2 yr old were 14.3%r and 2.8% respectively. Also notable was t h e presence of neoplastic nodules in the livers of 1.0% of the population, prevalences rising steeply with a g e and locally attaining values of up to 3 0 % in 6+ yr old fish. Most of the samples were collected in September when flounder are resident in inshore feeding arcas. Using data from 9 sites, spatial and temporal (year-to-year) variation in disease occurrence was analysed statisti-cally using log-linear models which incorporated possible effects of length, a g e and sex. Year-to-year variation showed little correspondence among the 3 diseases, but their spatial distributions showed striking similarities. Lymphocystis and skin ulcers were associated in individual fish. The observed variation in disease prevalence showed no significant correlation with condition factor of the fish or with concentrations of contaminants in sediments or tissues. However, disease preva-l e n c e ~ at different sites showed a strong positive correlation with fishing activity (possibly indi-cating an effect of damage by fishing gear) and appeared also to be positively related to salinity. When only strictly marine sites were considered, a relationship with pollution could not be ruled out. Additional data collected in February-April at offshore sites indicated that disease preva-l e n c e ~ were generally higher at this time of the year, which corresponds to the spawning period of the populations studied. This trend was particularly pronounced for liver neoplasms, and might be partly related to a low condition factor resulting from spawning activities. In view of the different aetiologies of the 3 diseases, the similarities in spatial patterns indicate the existence of 1 general underlying mechanism of disease causation, perhaps acting through immunosuppressive effects. However, age-related migration appeared to explaln some aspects of the spatial pattern of liver neoplasms, whereas it was less important in the case of epidermal diseases, which develop more rapidly. On the basis of the findings of this study, the possible contribution of pollution to disease prevalence cannot be adequately assessed due to the interfering effects of other factors (salinity, fishing activity, migration and spawning). Disease causation appears to be con~plex, and it may be that effects of pollution interact with those of other factors to produce observed, spatial patterns.
During 3 research cruises in the southern North Sea between 1986 and 1988 dab Limanda limanda L. were examined for liver anomalies. Macroscopic and Microscopic descriptions include different color irregularities and neoplastic changes. The frequency of the various liver lesions observed were calculated, the influence of fish length, sex, and season on their occurrence studied, and their geographical distribution was documented. It is discussed whether examination of dab livers is a suitable additional method in biological effect monitoring programs.
Tracie Eileen Bunton received a DVM degree from Michigan State University in 1977. After practicing small animal medicine for a year, she completed a combined Comparative Pathology residency/Ph.D. program in 1982 at the University of California at Davis. Her residency training included 3 yr at the California Regional Primate Research Center and, for her thesis, she studied the effects of glucocorticosteroids on lung development in the fetal rhesus macaque.
Dr. Bunton spent 1982-1984 as an Assistant Professor at Michigan State University, becoming a diplomate of the American College of Veterinary Pathologists in 1983. In 1984 she joined the faculty of the Division of Comparative Medicine at the Johns Hopkins University School of Medicine, where she is now an Associate Professor, with adjunct faculty positions in the Department of Pathology and the Graduate Program in Cellular and Molecular Medicine.
Dr. Bunton's primary research interest is on mechanisms of toxicity and carcinogenesis in fish related to environmental contamination and on the development of alternative models.
Piscine macrophage aggregates (MAs), alternatively known as melano-macrophage centers (MMC), are focal accumulations of macrophages usually containing the pigments hemosiderin, lipofuscin and ceroid, and melanin. The structures are not confined to fish and have been observed in other poikilothermic vertebrates. The aggregations are most commonly present in the spleen, pronephros, mesonephros and liver but may be found in other organs, especially in relation to inflammation. In the lower fishes (Agnatha, Chondrichthyes) the pigmented cells tend to be solitary or in small (<30 cells/aggregation), irregularly shaped aggregations primarily in hepatic tissue. In the Osteichthyes, greater numbers of cells and aggregates are present than in the lower fishes. These aggregates are more nodular, and they occur more commonly in the spleen and kidney rather than the liver.
A 5-week exposure of adult medaka (Oryzias latipes) to diethylnitrosamine (DEN. 50 ppm in aquarium water), followed by recovery, in clean water, results in hepatocellular carcinoma 12–16 weeks after initiation of exposure. Serial (weekly) morphometric evaluations estimated alterations within the liver. Enzyme histochemistry, marked putatively carcinogen-initiated hepatocytes in microscopic foci. Foci progressed, with time, to areas and nodules. The continued finding of hepatocyte necrosis in nonmarked populations and expansion of marked populations suggests resistance and growth of previously initiated cells. Cells marked by γ-glutamyl transpeptidase, quinone oxidoreductase (DT-diaphorase) and glucose-6-phosphate dehydrogenase were found in resultant neoplusms, suggesting linkage of focal phenotypes to eventual carcinomas. Tinctorial alterations and enzymic properties were compared in individual foci, areas, nodules and tumors. Biochemical verification of enhanced activity of γ-glutamyl transpeptidase and DT-diaphorase were obtained.
A carcinogenicity- and mutagenicity-directed fractionation approach was used to identify the carcinogenic compounds in contaminated sediments that are putatively responsible for the high prevalence of tumors in bottom-dwelling fish from Hamilton Harbour, Ontario. Mutagenic activity was detected with Ames tester strains (TA98, TA100) in relatively nonpolar fractions of sediment extract containing PAHs and nitrogen-containing aromatic compounds (NCACs). These fractions were also carcinogenic in an in vivo carcinogenicity bioassay with rainbow trout (Oncorhynchus mykiss). When a more polar extract fraction was tested for mutagenicity and carcinogenicity, weak mutagenic activity was detected with an O-acetyltransferase-enriched Ames tester strain (YG1024), and weak carcinogenic activity was detected in the rainbow trout assay. These data indicate that PAHs in contaminated Hamilton Harbour sediments are potent fish carcinogens, but it is also evident that other organic compounds in the sediment, such as NCACs and nitroarenes, may contribute to carcinogenicity.
Effluent from sewage-treatment works entering British rivers contains an estrogenic chemical, or mixture of chemicals, that stimulates vitellogenin synthesis in male fish. If the effluent constitutes a significant proportion of the flow of the river, lengthy stretches of entire rivers can be estrogenic to fish. The chemical, or chemicals, responsible for this feminizing effect have not yet been identified. However, many man-made chemicals known to be estrogenic to fish (and other vertebrates) are present in effluent, although which of these, if any, is responsible for the effects noted when caged fish are placed in rivers is unclear presently. In laboratory studies, exposure to estrogenic alkylphenolic chemicals caused a reduction in the rate of testicular development in trout undergoing sexual maturation.
Morphological alterations of the liver of zebra fish (Brachydanio rerio) following prolonged exposure to 0.1, 1 and 5 mg · l−1 4-nitrophenol (4-NP) were investigated by means of light and electron microscopy. Based on marked sexual dimorphism in control animals, liver recactions were both sex- and dose-dependent. Whereas at 0.1 mg·1−1 only minor changes could be revealed, there were numerous structural modifications at 1 mg·1−1: Whereas male fish primarily react with a proliferation of smooth endoplasmic reticulum, female fish display a high degree of fenetrastion within cisternate of the rough endoplasmic reticulum. Both sexes exhibit infiltration of macrophages and lymphocytes, an increase in the number of binucleate cells, of lysosomes, autophagosomes and myelinated bodies, but a depletion of hepatic glycogen. At 5 mg·1−1, deformations of the nuclear membrane and partial lysis of mitochondria could be observed. At 1 and 5 mg·1−1 4-NP about 25% of the animals investigated showed symptoms of degenerative transformations of the liver tissue into huge multinucleate cell masses with completely different ultrastructure.The evaluation of the results leads us to the conclusion that for toxicological studies on the effects of ‘priority pollutants’ histological and cytological investigations should be incorporated into an integrated methodological scheme.
Two toxicity experiments were carried out with the β-isomer of hexachlorocyclohexane (β-HCH) in the Japanese ricefish (medaka, Oryzias latipes). The experiments were started with freshly fertilized eggs (Exp. 1) or with young fish (1 mth post hatching) (Exp. II). The concentration of β-HCH ranged from 0.032–1.0 mg/l tank water. After 1 and 3 mth histopathological examination was carried out, which revealed development of testis-ova (intersexuality, hermaphroditism) in males and induction of vitellogenesis in either sex after 3 mth. These features are characteristic of estrogen-like activity. In addition, after 1 and 3 mth heterotopic development of adipose tissue (lipomatosis) was observed in parenchymal organs (liver, kidney, testis) which in the liver was associated with multilocular dilations of the interstitium (spongiotic edema) in Exp. I. In this experiment, the vacuolation of liver cells was also increased, which electronmicroscopically appeared to be due to accumulation of glycogen. In the thyroid follicles the epithelial cells showed hypertrophy and the colloid content was diminished; moreover, the number of thyrotropic hormone-producing cells in the pituitary was increased. These observations are indicative of a high level of activity of the thyroid. In the kidneys accumulation of amorphous eosinophilic precipitate in the glomeruli (glomerular hyalinosis) was prominent, and a similar precipitate could be found around the liver sinusoids and the splenic capsule; the nature of this precipitate could not be determined histochemically and electronmicroscopically. It is concluded that β-HCH has multiple toxic effects in medakas, some of which yield evidence for an estrogen-like activity, as is also found in guppies and rodents. Exposure for 1 or 3 mth in both experiments produced no-effect concentrations of 0.056 and 0.1 mg β-HCH/1 respectively.
Cytological changes during progression of experimentally induced hepatic neoplasia in fishes were reviewed with emphasis on recent findings in Cyprinodon variegatus and Oryzias latipes. Hepatocytes are particularly sensitive to toxic changes during early phases of response to carcinogens reflecting both lethal and sublethal alterations. In these degenerative lesions, enzyme histochemical studies reveal marked deficiency of glucose-6-phosphate dehydrogenase, glucose-6-phosphatase and adenosine triphosphatase. Surviving hepatocytes are either enlarged, encircled by cells with small nucleus to cytoplasm ratios, and have altered nuclear morphology suggestive of an inability to divide, or are smaller, apparently rapidly dividing, and have basophilic cytoplasm. In both species, development of spongiosis hepatis occurred following cytotoxic phases. This lesion apparently provides abundant space for cellular remodeling during neoplastic progression leading to eventual multinodular change. Foci of altered hepatocytes included basophilic, eosinophilic (both species) and clear cell (Cyprinodon variegatus only). Enzyme alterations preceded other tinctorial, morphologic alterations and were seen in cells composing foci and tumors, suggesting lineage of phenotypic alteration. Cytologic changes within other resident cell populations during neoplastic progression were reviewed.
Freeze-drying and glycolmethacrylate embedment (FDGE), without prior fixation and chemical dehydration, has proven useful in the study of the progression of lesions associated with diethylnitrosamine-induced liver neoplasia in medaka (Oryzias latipes). Localization of specific enzymes including acid phosphatase (ACP), alkaline phosphatase (ALKP), gamma-glutamyl transpeptidase (GGT), uridine diphosphate glucose dehydrogenase (UDPGDH), quinone oxidoreductase or DT diaphorase (DTD), and glucose-6-phosphate dehydrogenase (G6PDH) was achieved histochemically and correlated, in serial sections, with the conventional histologic stain, hematoxylin and eosin. Phenotypes of enzyme-altered foci and subsequent lesions in the promotion and progression of hepatocarcinogenesis were determined. GGT and G6PDH marked toxic and inflammatory phases respectively, while GGT, DTD, ALKP, ACP, G6PDH and UDPGDH proved useful in differentiating phenotypically altered hepatocytes during the later stages of neoplastic development. FDGE permitted the evaluation of multiple tissue markers in serial sections. limited the development of diffusion artifacts, produced higher resolution than cryostat sections and proved nearly as good as conventional light microscopy for routine survey. Integration of morphologic and biochemical data within microscopic lesions in a single small liver has been achieved.
Alanine aminotransferase (GPT) activity was measured in plasma and liver from rainbow trout fed two commercial fish diets.Plasma enzyme activities for the two groups of fish were significantly different at 3, 6, 12 and 18 hr after treatment with CCl4 at 1.0 ml/kg.No correlation was found between the degree of hepatocellular damage and elevated plasma GPT activity or the diet.Central vein necrosis was not evident in either dietary group of CCl4-treated trout.Dietary constituents and contaminants are discussed as potential variables in this study.
The toxicity of two chlorinated hydrocarbons, carbon tetrachloride (CCl4) and chloroform (CHCl3) was studied in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes. The aim was to study the cytotoxicity of these two chemicals in vitro in fish hepatocytes and to evaluate the sensitivity of the model system in comparison to mammalian systems. Both chemicals showed a steep dose response in this system with CCl4 being five times more toxic than CHCl3 measured as LDH release. Glutathione levels were rapidly depleted in the presence of CCl4 and CHCl3. There was a difference, however, in the pattern of GSH depletion. CCl4-induced GSH depletion followed the release of LDH, while CHCl3 caused a significant decrease in the GSH content prior to LDH release. The cytochrome P450 inhibitor, SKF-525A (12 μM) decreased the toxicity of 5 mM CCl4 and of doses ranging from 10 to 25 mM CHCl3. The addition of the antioxidant DPPD (20 μM) decreased the toxicity of both CCl4 and CHCl3. The protection was more pronounced with CHCl3 than with CCl4. When the time course of GSH depletion with EC50 concentrations of each chemical was studied, DPPD effectively protected the cells from CHCl3-induced GSH depletion, while no significant maintenance of GSH was seen in cells treated with DPPD and CCl4. The relationship between DNA single strandbreaks (SSB) and cytotoxicity may provide some insight into potential carcinogenicity of non-genotoxic chemicals. In cells treated with CHCl3, DNA SSB were seen only concommitant with high toxicity. With CCl4, however, DNA SSB were seen in the absence of LDH release at 2.5 mM. This study indicates that the toxicity of CCl4 and CHCl3 in trout hepatocytes is due to metabolism and the formation of free radicals at doses causing low toxicity and to a combination of metabolism dependency and solvent effect at doses causing high toxicity. This study also indicates that there are differences in the mechanisms of toxicity of CHCl3 and CCl4 in rainbow trout hepatocytes. Furthermore, the model system based on primary cultures of rainbow trout hepatocytes is a sensitive tool in toxicological studies and is comparable to model systems based on mammalian hepatocytes.
Female freshwater eels injected with estradiol-17β (E2) for 15–78 days appear paler and secrete more mucus than controls. The resulting strongly opalescent blood plasma indicates that vitellogenin synthesis occurs in the liver, which shows a significant hypertrophy, an increased vacuolization (lipid material) and glycogen depletion. Plasma sodium is lowered, but calcium levels are considerably increased. The gonosomatic index increases (0.92±0.1 to a maximum of 2.21). Oocytes are enlarged, but the incorporation of vitellogenin remains discrete. Gonadotrophs (GTH cells), small and scarcely visible in the pituitary of control eels, are hypertrophied and contain numerous glycoprotein granules after E2-administration. E2 may act on the pituitary and/or hypothalamus via a positive feedback to induce gonadotrophin (GTH) synthesis; GTH release seems to be very limited as indicated by the ovarian response. The differentiation of GTH cells in eels treated with fish pituitary extracts is most probably due to secretion of E2 by the ovary, which reacts on the pituitary. Various hypotheses are considered to explain the low GTH release.
Thyrotrophs, somatotrophs and prolactin cells of the pituitary are stimulated. In the pars intermedia, MSH and PAS-positive cells appear less active. A possible antidopaminergic effect of E2 is discussed.
E2 administration constitutes a simple and economic technique to induce the synthesis of GTH and will facilitate the biochemical and biological study of the latter hormone in eels.
A new type of perisinusoidal cell containing numerous microfilaments is described for the first time. It is found in abundance in the livers of both marine and freshwater fish. These perisinusoidal cells are situated within the space of Disse and adhere firmly through desmosomes both to sinusoidal endothelial cells and to hepatocytes. The cytoplasmic microfilaments are striking and make these cells readily distinguishable from the perisinusoidal fat-storing cells of Ito. Although the function of these cells is not known, the observations presented here suggest that they may provide a supportive framework within the liver.
Carbon Tetrachloride-Induced Retention of Sulfobromophthalein in the Plasma of Rainbow Trout. Gingerich, W. H., Weber, L. J., and Larson, R. E. (1978). Toxicol. Appl. Pharmacol.43, 147–158. Carbon tetrachloride was evaluated as a potential hepatotoxic agent in rainbow trout. Plasma half-lives of sulfobromophthalein (BSP) increased in proportion to the dose of CCl4 administered, and histological examination of liver sections indicated that morphological damage, including necrosis of hepatocytes surrounding central veins, also had occurred. High plasma hemoglobin concentrations, hemoglobinuria, and a significant increase in relative body weight were observed in trout following intoxication. To exclude the possibility that plasma BSP clearance in treated fish had been impaired by high plasma hemoglobin concentrations, plasma BSP disappearance studies were conducted in two groups of fish following prolonged infusion of either bilirubin 40 μg/kg/min) or hemoglobin (250 μg/kg/min). In these studies, plasma BSP disappearance was not affected by high plasma concentrations of either compound, and it was either bilirubin (40 μg/kg/min) or hemoglobin (250 μg/kg/min). In these studies, plasma BSP disappearance was not affected by high plasma concentrations of either compound, and it was therefore concluded that the retention of BSP in plasma of fish treated with CCl4 could not be explained by attendant intravascular hemolysis. Carbon tetrachloride appears to serve as a useful model hepatotoxic agent in rainbow trout and further, retention of BSP in the plasma may be a useful index by which to evaluate liver dysfunction in this fish.
Uptake, distribution, and effects of CCl4 were studied in rainbow trout. Carbon tetrachloride (1 ml/kg, ip) produced 5- to 10-fold increases in serum GOT, GPT, and ICD activities, whereas exposure of trout to CCl4 in the tank water (1–80 mg/liter) produced neither mortality nor significant changes in enzyme activities. CCl4 residue(s) appeared highest in concentration in the adipose tissue, followed by liver, brain, and spleen, and was lowest in gill regardless of the administration route. The elimination rates of 14C residue(s) from the tissue samples were most rapid in muscle () and relatively prolonged for liver (). Maximum liver concentrations of 14C residue(s) were reached at 2 hr by either ip (1 ml/kg) or water exposure (80 mg/liter) and were 4.8 μmol/g and 0.75 μmol/g, respectively. No increase in liver triglyceride (TG) concentrations were noted at liver CCl4 concentrations that have been associated with increased TG levels in the rat. Histological examination of tissues revealed varying degrees of liver and splenic necrosis 6 hr after administration of CCl4.
In the normal carp livers, Ito cells (fat-storing cells) could be identified with light and electron microscope within the hepatic sinusoidal wall and actually in the Disse's space, but they were almost lacking lipid droplets in the cytoplasm (empty Ito cells) in contrast to the majority of vertebrate species. After administration of large doses of vitamin A, fat droplets appeared in hypertrophic cytoplasm of enlarged Ito cells, and their size and number increased roughly in proportion to the amounts of vitamin A administered. This evidence was thought to demonstrate that the administration of large doses of vitamin A could convert empty Ito cells into lipid containing ones and further suggested that excess vitamin A administered might be stored in newly prepared lipid droplets of the Ito cells of the liver. In experimentally hypervitaminotic carp, hypertrophic Ito cells showed proliferation of fine filaments in addition to the accumulation of lipid droplets. The correlation between perisinusoidal fibrogenesis in the hepatic lobule and proliferation of cytoplasmic filaments in the Ito cells was discussed.
Liver cytotoxic alterations of adult medaka (Oryzias latipes) following short-term bath exposure (48 hr) to 500 mg/L diethylnitrosamine (DEN) were studied (days 3-21) by electron microscopy and cytochemistry. Control medaka displayed hepatic sexual dimorphism as described for other sexually active fish. Following DEN exposure, decreased glycogen stores with loss of cellular compartmentation obscured sexual dimorphism. A spectrum of organelle alterations, previously not reported in livers of fish, was seen. Early changes in hepatocytes included: nuclear lipid inclusions, nucleolar changes, decreased amounts of granular endoplasmic reticulum (GER), increased fractionation and steatosis of GER, proliferation of smooth ER and lysosomes, reduction in number and content of particulate lipoproteins and vitellogenin in Golgi vesicles, and reduction in number and staining intensity of peroxisomes. At day 14 and/or 21, partial to complete reversal of the above alterations indicated hepatic recovery, and fewer necrotic cells were seen at day 21 versus day 14. Lesions that did not resolve during this study were altered mitochondria and areas of spongiosis hepatis that developed at day 8 and continued to increase throughout the study. Infiltration of lymphocytes, granulocytes, and large numbers of macrophages were late changes. The description, timing, and duration of lesions are of value for consideration as biomarkers of exposure and effect in aquatic toxicology.
The role of a putative liver stem cell in liver regeneration and carcinogenesis is reviewed. There is increasing evidence that there is a liver stem cell that has the capacity to differentiate into parenchymal hepatocytes or into bile ductular cells. These stem cells may be activated to proliferate after severe liver injury or exposure to hepatocarcinogens. They are not activated by moderate liver injury, which is repaired by proliferation of mature hepatocytes. Exposure to most chemical hepatocarcinogens results in proliferation of a small morphologically indistinct cell population termed "oval cells." These cells have been shown to have the capacity to differentiate into hepatocytes or into ductular cells. The origin of these cells appears to be from transition duct cells, but there is also evidence of an even less mature periportal liver stem cell. Study of the development of these cells during carcinogenesis indicates that liver cancer arises from oval cells by aberrant differentiation of stem cells.
Spongiosis hepatis (SH), first reported as a distinct lesion associated with certain forms of hepatic neoplasia in rats, has also been induced with chemicals, in a predictable fashion, in small teleost fishes being studied as carcinogenesis research models. The sheepshead minnow (Cyprinodon variegatus), exposed to N-nitrosodiethylamine (DENA) in sea water, provided the model for this study. The fish developed SH and presented a spectrum of developmental or progressive stages of the lesion over a 140 week holding period following a 6 week exposure to / 57 mg/L DENA. The origin of SH in the fish model is homologous to that in the rat model, both species having the perisinusoidal cell (stellate cells of Ito) in the space of Disse as the cell of origin. Light (LM) and electron microscopy (EM) studies characterized the different pathogenetic stages of SH in liver of the sheepshead minnow and revealed a possible late transition of SH to putative polymorphic cell neoplasms. The possible preneoplastic or neoplastic nature of SH from its time of origin in chemically exposed fish to time of appearance of associated presumptive neoplasms is discussed. SH may be a bioindicator of exposure to certain chemicals in some vertebrate species, from fishes to mammals.
The effect of short-term exposure (48 hr) of 14-day-old medaka (Oryzias latipes) to diethylnitrosamine (DEN) was tested on 4 groups of fish at 0, 100, 200, and 400 mg/L, with interim sacrifices of up to 6 months post exposure. Many of the lesions seen were similar to those seen in other fish DEN carcinogenicity studies using longer term, adult exposures, including a cytotoxic phase with cystic degeneration of the hepatic parenchyma, cellular vacuolation, globular acidophilic cytoplasmic inclusions, spongiosis hepatis, foci of cellular alteration, adenomas, and cholangiomas. There were also several proliferative lesions of uncertain origin which occurred throughout the study. There appeared to be a direct relationship between exposure level and the incidence and severity of the lesions seen, including aggressiveness and invasiveness of the neoplasms seen. However, there was a possible difference from other studies in the types of neoplasms seen with this protocol, which may be related to age at exposure, exposure level, duration of exposure, or a combination of these factors. Similarities in lesions seen in this study and in rodent and other fish studies is an indication of promise for the use of this model in carcinogenicity testing. Differences indicate the variety of ways the fish liver can respond to toxic injury, and lend flexibility to the model.
Quantitative evaluation of altered hepatocellular foci (AHF), followed by stereological analysis was performed on standard hematoxylin and eosin-stained liver sections from control Fischer 344 (F344) rats of both sexes from seven 2-yr carcinogenicity studies conducted by the National Toxicology Program (NTP). Liver samples were collected at 6, 9, 12, 15, 18, and/or 24 months on study. Although AHF had a broad spectrum of morphological features, they could be classified into the following 5 types using previously published criteria: basophilic, eosinophilic, clear, vacuolated, mixed cell foci. Approximately 50% of the animals had foci at 6 months, and the incidence reached nearly 100% at 15 months in both sexes. The number, size and volume fraction of AHF increased with age in both sexes; these changes were most evident for basophilic and clear cell foci. The number of basophilic foci was significantly greater in females than in males while clear cell foci were more numerous in males. This sex difference was observed at each time point. Mean number of all types of AHF in males and females at 24 months was 547 and 460 per cubic centimeter of liver, respectively. Despite the high incidence of AHF in control rats, the incidence of hepatocellular neoplasms is low. The implication is that most foci do not progress to neoplasia in control F344 rats used in 2-yr studies.
Histochemical markers are important for the early detection of chemically initiated neoplasia in experimental animal studies. The marker, iron resistance, was evaluated in the Shasta strain of rainbow trout (Salmo gairdneri). Twenty-one-day-old trout embryos were exposed to 100 ppm aqueous N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 30 min in a static water bath. Fish were fed a semipurified diet, and sampled monthly from the 4th to the 9th month. Two days before sampling, fish were iron-loaded with a single ip dose of 0.30 mg iron dextran/100 g body weight. Livers and kidneys were conventionally processed to paraffin sections for iron, or hematoxylin and eosin (H&E) staining. Normal hepatocytes accumulated iron in pericanalicular locations, but in hepatocytes from carcinogen-altered foci and tumors, iron staining was clearly reduced or absent. Normal renal tubule cells exhibited slight to moderate iron staining, while those from nephroblastoma were iron resistant. These results establish iron resistance as a property of preneoplastic and neoplastic trout hepatocytes and nephroblastoma cells for the first time. Iron resistance may offer a practical histochemical marker in experimental fish models of hepatocellular carcinoma and nephroblastoma.
Tests of statistical hypotheses concerning treatment effect on the development of hepatocellular foci can be carried out directly on two-dimensional observations made on histologic sections or on estimates of the density and volume of foci in three dimensions. Inferences about differences in the density or size of foci from tests based on two-dimensional observations, however, can be misleading. This is because both the number of focus cross-sections observed in a tissue section and the percent area occupied by foci can be expressed in terms of the number of foci per unit volume of liver tissue and the mean focus size. As a consequence, a treatment difference may be caused by a difference in the density of foci, their average size, or both. Of more serious concern is the possibility that failure to detect a treatment effect may occur not only when there is no treatment effect but also when the density and size of foci differ between treatments in such a way that their product is unchanged. This can happen if the effect of treatment is to increase the number of foci and decrease their average size, or vice versa. A similar difficulty of interpretation is associated with hypothesis tests based on average focus cross-section area. Tests based on estimates of the number of foci per unit volume and mean focus volume allow direct inference about the quantities of interest, but these estimates are unstable because they have large variances. Empirical estimates of statistical power for the Wilcoxon rank sum test and the t-test from data on control rats suggest power may be limited in experiments with group sizes of ten and low observed numbers of focus cross-sections. If hypothesis tests based on estimates of the density and size of foci are to form the basis for a bioassay, then the power of statistical tests used to identify treatment effects should be investigated.
Hepatic stroma and parenchyma with its component cell types were quantitatively described in adult male and female actively-spawning 5-year-old rainbow trout (Salmo gairdneri, Richardson). Point-count morphometry of glycol methacrylate sections estimated volume compartments for stroma and parenchyma. Veins composed 85% of the stroma while arteries and bile ducts occupied approximately 6-7% each. Parenchyma accounted for 95% of hepatic volume. Point-count morphometry of transmission electron micrographs estimated volume compartments as well as numerical and surface density measurements for parenchymal components. Within the hepatic parenchymal compartment, hepatocytes occupied 85% and showed significant sex differences. Female hepatocytes were significantly more numerous but were smaller, only 60% of the volume of male hepatocytes. Since hepatocyte nuclear volume was equal in both sexes, differences were due to reduced cytoplasmic volume in females. Perisinusoidal macrophages of females occupied larger volumes of their respective parenchymal compartments, and their larger mean cytoplasmic volumes suggested activation. Biliary epithelial cells of preductules and ductules were numerous. Ratios of numerical density of hepatocytes to biliary epithelial cells were consistent with a tubular arrangement of hepatocytes. Factors possibly mediating the sexual dimorphism are discussed.
The intrahepatic biliary system was studied in the rainbow trout (Salmo gairdneri), a teleost known to form liver neoplasms after exposure to various carcinogens. Normal adults (N = 25) were examined using light microscopic, enzyme histochemical, and transmission and scanning electron microscopic methods. In light micrographs, longitudinal arrays of hepatocytes appeared as double rows incompletely divided by elongated darkly stained cells. Electron micrographs showed tubules of five to nine pyramidally shaped hepatocytes with their apices directed toward a central biliary passageway and their bases directed toward sinusoids. Sequentially, beginning with hepatocytes, biliary passageways included canaliculi, preductules, ductules, and ducts. Canaliculi were short and joined transitional passageways (preductules) formed by junctional complexes between plasma membranes of hepatocytes and small, electron-dense cells with a high nuclear to cytoplasmic ratio. Ductules, completely lined by biliary epithelial cells, occupied central regions of hepatic tubules. Relatively elongated, ductular cells were intimately associated with surrounding hepatocytes, separated from them by only a thin extracellular space devoid of a basal lamina. Epithelium of bile ducts included cuboidal through mucus-laden columnar cells, surrounded by basal lamina and, in larger ducts, by fibroblasts, smooth muscle cells, and a capillary plexus. Bile ducts and hepatic arterioles, but not venules, were distributed together. The ultrastructure of biliary epithelium, periductular, and periductal cells is presented.
The livers of 26 adult male and female trout were studied histochemically. G6Pase activity was always found to be heterotopically distributed with a constant maximum in the periportal area. In many cases the glycogen content and the activity of phosphorylase predominated in the periportal zone as well. Maximum activity of glucose-6-phosphate-dehydrogenase and malic enzyme, however, could be demonstrated preferentially in the perivenous area. Lactate dehydrogenase, succinate dehydrogenase, alcohol dehydrogenase, acid phosphatase and beta-glucuronidase were found equally in all liver cells. 3-Hydroxybutyrate dehydrogenase was absent. Thus, the principles of metabolic zonation have been established in trout liver, the architecture of which differs essentially from that of mammals. The course of the terminal afferent and efferent vessels is the decisive factor for the heterotopic localization of functional units rather than the tubular or plate-forming arrangement of the hepatocytes.
The architectural arrangement and selected histochemical properties of hepatocytes in the rainbow trout (Salmo gairdneri Richardson) were examined. Light and transmission electron microscopic (TEM) examination following fixation by portal venous perfusion revealed a tubular arrangement of hepatocytes. Lobules, as defined in the adult mammal, were absent. Biliary epithelial cells associated with bile preductules and ductules were a prominent feature of trout liver. Patterns and location of reaction products for glucose-6-phosphatase (G-6-Pase), glucose-6-phosphate dehydrogenase (G-6-PDH), and magnesium-dependent adenosine triphosphatase (ATPase), enzymes preferentially distributed in mammalian liver, were demonstrated in trout liver. A slightly heavier staining pattern for G-6-Pase was seen around presumptive portal venules but all other enzyme reaction patterns were uniform throughout the liver parenchyma. Following ATPase localization, four sizes of biliary passageways (canaliculi, bile preductules, ductules, and ducts) were visualized. Maximum glycogen retention was achieved with freeze-drying and glycolmethacrylate embedding and with this method intense, uniform glycogen staining was observed in all areas of the liver. Companion TEM examinations revealed large depots of glycogen within hepatocytes. The results are important for interpretation and description of the effects of toxic/carcinogenic alteration on trout liver.