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Tumor Induction by Carcinogenic Agents in Aquarium Fish
Abstract
The effects of nine carcinogens on 1,220 guppies [Poecilea reticulata (Lebistes reticulatus)] and 40 zebra fish (Danio rerio) were studied. Multiple exposure techniques included skin application, im and ip injections, feeding, implantation in pellets, and dissolving of the compound in the aquarium water. 7-12-Dimethylbenz[a]anthracene, 3-methylcholanthrene, and benzidine produced no tumors in the fish. N-2-Fluorenylacetamide, omicron-aminoazotoluene, 4-dimethylaminoazobenzene, diethylnitrosamine, and dimethylnitrosamine induced tumors in the livers of some of the fish. These neoplasms included cholangiomas, hepatoadenomas, cholangiocarcinomas, and hepatocellular cancers. Nitrosomorpholine caused not only hepatic tumors in guppies and zebra fish but also intestinal adenocarcinomas and poorly differentiated connective-tissue lesions in the abdominal cavities of zebra fish. Experimental induction of tumors in aquarium fish offers wide possibilities for comparative cancer research. Fish are a suitable model for the testing of compounds for carcinogenic activity and for the screening of environmental carcinogens.
... Extensive fibrosis in hepatic parenchyma and venoocclusive disease in the centrolobular and hepatic veins were also present (Hendricks et ai., 1981a). Kidney: Thickened glomerular basement membranes at higher doses (Hendricks et aI., 1981a Pliss and Khudoley, 1975). Liver tumors (Halver, 1967). ...
... Liver tumors (Halver, 1967). Liver tumors ( Pliss and Khudoley, 1975). Adenomatous hyperplasia in liver ( Kimura and Kubota, 1972) and liver tumors (Khudoley, 1972;Pliss and Khudoley, 1975). ...
... Liver tumors ( Pliss and Khudoley, 1975). Adenomatous hyperplasia in liver ( Kimura and Kubota, 1972) and liver tumors (Khudoley, 1972;Pliss and Khudoley, 1975). Liver tumors (Halver, 1967). ...
... The zebrafish embryos (<96 hours old) are considered an alternative model as it conforms to the 3Rs of animal experimentation [10]. Since zebrafish embryos easily absorb molecules inserted into their water, the first studies with the species were already based on the teratogenic effects in embryos previously exposed to chemical substances [11,12]. Since then, zebrafish have been used as a tool in toxicological analysis [12]. ...
Tel.: +55-19-997866505 (M.A.M.M.). Abstract: Anthropogenic interventions have had a compromising effect on environmental health, intensifying the degradation of ecosystems, and the quantity of chemical pollutants released into nature. Therefore, research areas within the scope of environmental assessments and monitoring such as ecotoxicology have contributed to the determination of the toxic potential of contaminants. A small cyprinid known as the zebrafish (Danio rerio), the use of which has exponentially grown, is an alternative vertebrate model for scientific research, mainly in the assessment of environmental risks. The species exhibits several advantages for breeding in a laboratory, in addition to presenting multi-biomarkers of environmental toxicity. Thus, this review aims to present the main characteristics and advantages of working with this species, as well as show studies related to ecotoxicology involving biomarkers of toxicity in zebrafish. The results show a progressive trend towards employing the species in environmental risk analyses, it is an increasingly recommended species in the assessment of the toxicity level of a range of chemical pollutants. The development of future technologies must contribute to scientific advancement, rendering the potential application of this model organism an even more widespread one, which will certainly help in bridging knowledge gaps in various areas of study. HIGHLIGHTS • Biomarkers of toxicity in zebrafish. • Toxicological screening with the embryonic, larval, and adult stages of zebrafish. • Scientific advancements in the assessment of the effects of environmental contaminants. Braga, A.P.A.; et al. 2
... The zebrafish embryos (<96 hours old) are considered an alternative model as it conforms to the 3Rs of animal experimentation [10]. Since zebrafish embryos easily absorb molecules inserted into their water, the first studies with the species were already based on the teratogenic effects in embryos previously exposed to chemical substances [11,12]. Since then, zebrafish have been used as a tool in toxicological analysis [12]. ...
... Previously, researchers appreciated the relative ease of treating fish with carcinogens because the chemicals can be dissolved in water and the animals can be exposed for longer periods. When exposing zebrafish to different compounds [e.g., 7,12-dimethylbenz(a)anthracene, Nnitrosodimethylamine, and N-nitrosodiethylamine], mainly liver and intestinal tumors were observed [5][6][7] . More in recent times, similarly but more extensive studies showed that N-nitrosodiethylamine primarily induces liver and pancreas carcinomas (8), and Nnitrosodimethylamine induces only liver tumors 9 . ...
The zebra fish has developed into an important model organism in biomedical research over the last two decades. Although the main focus of zebra fish research has habitually been on developmental biology, observing zebra fish in the lab led to the identification of diseases similar to humans, such as cancer, which subsequently became a subject for study. As a result, about more than 50 articles have been published since 2000 in which zebra fish were used as a cancer model. Strategies used include carcinogenic treatments, transplantation of mammalian cancer cells, forward genetic screens for proliferation or genomic instability, reverse genetic target-selected mutagenesis to inactivate known tumor suppressor genes, and the generation of transgenics to express human oncogenes. Zebra fish found to develop almost all type of tumor known from human, with similar morphology and, accordance to gene expression array studies, equivalent signaling pathways. However, tumor incidences were found to be relatively low, although highly comparable between different mutants, and tumors develop late in life. In addition, tumor spectra are sometimes different when compared with mice and humans. However, the zebra fish model has created its own slot in cancer research, complementing existing models with its specific experimental advantages and characteristics. Examples of these are imaging of tumor progression in living fish by fluorescence, treatment with chemical compounds, and screening possibilities not only for chemical modifiers but also for genetic enhancers and suppressors. This review aims to provide a comprehensive overview of the state of the art of zebra fish as a model in cancer research.
... A single subcutaneous injection of 2-acetylaminofluorene caused liver tumors (hepatocellular tumors) in newborn male mice (Fujii 1991). Liver tumors were also observed following dietary administration of 2-acetylaminofluorene to male dogs (Allison et al. 1950) and to fish of both sexes (hepatocellular tumors or cholangiocarcinoma) (Pliss and Khudoley 1975) and following addition of 2-acetylaminofluorene to the tank water of fish of unspecified sex (hepatocellular adenoma or carcinoma) (James et al. 1994). In hamsters of both sexes, intratracheal instillation of 2-acetylamino fluorene caused urinary-bladder cancer (transitionalcell carcinoma) (Oyasu et al. 1973). ...
... Advantageously, zebrafish can endure treatments at a variety of chemical concentrations and durations. For instance, smaller doses, from 5 mM or less can be applied for up to 24 hours, while doses greater than 20 mM are to be applied for 8 hours or less [11]. The treatment of zebrafish with the mutagen 7,12-dimethylbenz(a) anthracene induces the broadest range of tumors, from epithelial tumors in intestines to mesenchymal tumors in blood vessels and lymphoid malignancies (Table 1) [12]. ...
The ability of zebrafish to faithfully recapitulate a variety of human cancers provides a unique in vivo system for drug identification and validation. Zebrafish models of human cancer generated through methodologies such as transgenesis, gene inactivation, transplantation, and carcinogenic induction have proven similar to their human counterparts both molecularly and pathologically. Suppression of cancer-relevant phenotypes provides opportunities to both identify and evaluate efficacious compounds using embryonic and adult zebrafish. After relevant compounds are selected, preclinical evaluation in mammalian models can occur, delivering lead compounds to human trials swiftly and rapidly. The advantages of in vivo imaging, large progeny, and rapid development that the zebrafish provides make it an attractive model to promote novel cancer drug discovery and reduce the hurdles and cost of clinical trials. This review explores the current methodologies to model human cancers in zebrafish, and how these cancer models have aided in formation of novel therapeutic hypotheses.
... Nevertheless, masses should be aspirated and examined cytologically to determine if they may be attributable to inflammation from infectious disease agents (bacteria, fungus, or metazoans) or a neoplastic lesion. This has some bearing on the prognosis and importance to other fish in a pond or aquarium system [21,22]. ...
... Advantageously, zebrafish can endure treatments at a variety of chemical concentrations and durations. For instance, smaller doses, from 5 mM or less can be applied for up to 24 hours, while doses greater than 20 mM are to be applied for 8 hours or less [11]. The treatment of zebrafish with the mutagen 7,12-dimethylbenz(a) anthracene induces the broadest range of tumors, from epithelial tumors in intestines to mesenchymal tumors in blood vessels and lymphoid malignancies (Table 1) [12]. ...
... A number of extrahepatic cancers were induced by DMBA in medaka, but the types were not specified 23 . However, the cancers were reportedly absent in guppy exposed to DMBA by intramuscular, intraperitoneal, or topical routes 24 . DMBA is a much more potent hepatocarcinogen in trout than B[a]P and produces tumours in kidney, swim bladder and stomach as well 25 . ...
The pollution of rivers and streams with chemical contaminants has become one of the most critical environmental problems. Fish living in a polluted water reservoir use the contaminated water to rinse their gills; this results in the deposition of polycyclic aromatic hydrocarbons (PAHs) in the fish body. Contamination of foodstuffs by heavy metals such as arsenic, cadmium, chromium, nickel and lead has poses a potential carcinogenic threat to humans. Arsenic and cadmium appear to be the most harmful to the fish. Several cancers in fish appear to be the result of exposure to different environmental pollutants/chemicals. High frequencies of liver and skin cancers in brown bullheads are associated with high concentrations of PAHs and some metals in the environmental sediments. Taking these facts in view, the present article gives the emphasis on the fish cancer caused by various environmental pollutants, suggesting that fish species are truly suffer from different cancers/tumours.
... The discussion remains unresolved if the increasing sea water pollution in the North Sea may produce a decreased resistance in the marine mammals (Breuer et al. 1988, Reijnders 1989) and so could predispose for neoplasia (Howard et al. 1983). In fish, however, there are reports that environmental carcinogenic substances may induce tumors of many kinds (Pliss & Khudoley 1975, Matsushima & Sugimura 1976, Sinnhuber et al. 1977, Couch & Courtney 1985, Couch & Harshbarger 1985, Hawkins et al. 1985. Nevertheless, the existing evidence does not permit a firm conclusion (Harshbarger 1977, MIX 1986. ...
Para las autoras es un logro presentar una obra que permite recopilar
los tres principales modelos biológicos usados en toxicología ambiental.
Estos conocimientos permitirán a nuestros estudiantes, profesionales y
personas interesadas en el tema adquirir una mayor compresión del uso
de estos modelos en las diferentes áreas como toxicología ambiental,
biotecnología y afines. Los usos de estos modelos son una herramienta
útil en diversas áreas del conocimiento científico, es de suma importancia
su difusión para aumentar el saber en las diferentes repercusiones que
tiene la contaminación ambiental en la salud humana.
Para tal fin, este libro ha sido conceptualmente dividido en tres capítulos;
en cada uno de los cuales se abordan las características generales de C.
elegans, H. vulgaris y D. rerio desde una perspectiva ecotoxicológica. Se
abarcan aspectos básicos como crecimiento, mantenimiento, anatomía,
avances logrados por medio de su estudio en diversas áreas de la
ciencia y finalmente su aplicabilidad en la investigación en el campo
de la toxicología ambiental, las novedades actuales y su impacto en la
salud. Además, se muestran consultas sistemáticas sobre estos modelos
con el fin de brindar información importante acerca de estos seres vivos
útiles para estudios toxicológicos.
Description
The book covers discussions of new concepts, methods and research results in aquatic toxicology and hazard assessment. This book is broken down into three sections: safety margins; environmental chemistry and fate modeling; and application of aquatic hazard assessment principles.
Tumor angiogenesis is the most crucial step in the progression of all types of cancers. Preexisted blood vessel vascularizes into new one through sprouting or intussusceptive (splitting) mechanism. This process would facilitate the growth in the size of tumors regulated by VEGF (vascular endothelial growth factor), leading to metastasis, which ultimately increases the severity of cancer. So, it is very important to suppress tumor angiogenesis before the situation gets worse in a cancer patient. A wide variety of in vitro and in vivo models have been used to study the process of tumor angiogenesis and metastasis of cancer. It has helped us to discover new drugs and to find novel therapies for cancer, including anti-angiogenic therapy. Mainly angiogenesis is traditionally modeled in rodents and chick embryo, but of late zebrafish is emerging as the preferred model due its several advantages over the other animals. Zebrafish (Danio rerio) serves as the ideal model to study the various cancers, since it is possible to induce tumor growth or suppression easily, when compared to the other animal models. Also, tumor xenograft model has been studied in zebrafish extensively using many human cancer cell lines. So, in this chapter, we have reviewed some literatures that appreciate zebrafish model to study tumor angiogenesis.KeywordsZebrafishAngiogenesisVEGFTumorXenograftAnti-angiogenic therapy
Zebrafish (Danio rerio) are a small‐bodied tropical, freshwater fish species originally from South Asia. The ease of care, year‐round prolific breeding, and transparent, external development have made these fish a popular model vertebrate for many fields of biology. Over the past 40 years, the development of methods for genetic and developmental manipulation have driven a rapid rise in their use in research. This review provides an orientation to the zebrafish as a model system, from its ecology and life history to its early use in research and adoption as a key genetic model for vertebrate development. Its current uses and key techniques that highlight future areas of development of the system are also discussed. Finally, an introduction to the essentials of zebrafish use and care are presented, highlighting the ease of adoption of this system for teaching and research use.
The zebrafish, Danio rerio (also referred to in the literature as Brachydanio rerio and the zebra danio), is currently emerging as an increasingly popular model of vertebrate embryonic development, gene function analysis, and mutagenesis (Fig. 20.1). Prior to the development of the zebrafish model in the 1970s, developmental geneticists relied on invertebrate models such as Drosophila melanogaster (fruit fly) and, more recently, Caenorhabditis elegans (nematode) for the investigation of early embryonic development. The prolific reproductive capacity of Drosophila and successful manipulation of its embryos combined to make embryo development and genetic analysis more practical in the laboratory. However, the application of this information to vertebrate embryonic development was limited. Since mouse embryos develop within an uterus and the African Clawed Frog (. Xenopus laevis) has a slow reproductive capacity, neither of these more popular vertebrate models possess the characteristics that made Drosophila such a practical model (Kahn, 1994). Because of its high fecundity and external fertilization, the zebrafish possessed the attributes of these existing models without their inherent drawbacks.
Zebrafish cancer models have provided critical insight into understanding the link between aberrant developmental pathways and tumorigenesis. The unique strengths of zebrafish as compared to other vertebrate model systems include the combination of fecundity, readily available and efficient transgenesis techniques, transparency that facilitates in vivo cell lineage tracing, and amenability for high-throughput applications. In addition to early embryo readouts, zebrafish can develop tumors at ages ranging from 2 weeks old to adulthood. Tumorigenesis is driven by genetically introducing oncogenes using selected promoter/tissue-specific expression, with either mosaic expression or with the generation of a stable transgenic line. Here, we detail a research pipeline to facilitate the study of human oncogenes in zebrafish systems. The goals of this approach are to identify conserved developmental pathways that may be critical for tumor development and to create platforms for testing novel therapies.
Here we describe the conditional zebrafish cancer toolbox, which allows for fine control of the expression of oncogenes or downregulation of tumor suppressors at the spatial and temporal level. Methods such as the Gal4/UAS or the Cre/lox systems paved the way to the development of elegant tumor models, which are now being used to study cancer cell biology, clonal evolution, identification of cancer stem cells and anti-cancer drug screening. Combination of these tools, as well as novel developments such as the promising genome editing system through CRISPR/Cas9 and clever application of light reactive proteins will enable the development of even more sophisticated zebrafish cancer models. Here, we introduce this growing toolbox of conditional transgenic approaches, discuss its current application in zebrafish cancer models and provide an outlook on future perspectives.
Liver and pancreatic cancers are amongst the leading causes of cancer death. In recent years, genetic and chemical approaches in zebrafish have elucidated cellular and molecular mechanisms of liver and pancreatic cancer formation and progression. In this chapter, we review the recent approaches and advances in the field to study both hepatocellular carcinomas and pancreatic cancer.
Teleosts provide sensitive animal models for use in carcinogenesis studies1–4. The Mt. Shasta strain of rainbow trout (Salmo gairdneri) is the most sensitive animal model used to study the tumorigenic effects of aflatoxin4. The brown bullhead (Ictalurus nebulosus) has emerged as a freshwater teleost species which is sensitive to induction of tumours by chemical carcinogen exposure, both experimentally in the laboratory and in the environment5–10. The brown bullhead, as well as other teleost species, may serve as sentinel organisms for the detection of biohaz-ardous material in the environment2–6. We report for the first time the light-and electron-microscopic features of experimentally induced hepatic tumours 9 months after 28-day exposure to an aqueous solution (100 ppm) of diethylnitrosamine (DENA) in 1 y old male and female brown bullheads. Lesions in the bullhead liver will be described in terms derived from the study of mammalian (rodent) hepatocarcinogenesis. Microscopic features of experimentally induced hepatic tumours will be compared with hepatic tumours found in feral brown bullheads previously sampled from the Black River, Lorain, Ohio. However, before descriptions and interpretations of the pattern and progression of lesions seen in bullhead liver during carcinogenesis can be made, knowledge of the normal architecture and component cell types is necessary.
Fundulus sp., a ubiquitous estuarine fish, was used in cytopathologic evaluations of the carcinogenic potential of incinerated ash from low-level radioactive waste, currently, disposed in the marine environment. We correlated the increased biosynthesis of Epoxide Hydrolase of liver microsomes with ultrastructural changes in livers of Fundulus sp., following exposure to non-radioactive and radioactive ash (7 ppm, radioisotope = S35, specific activity: 1410 Ci/mmol/kg ash). Fish were exposed to incinerated ash suspended in filtered artificial sea water in closed aerated/temperaturecontrolled marine tank systems. Macroscopic observation of animals exposed to radioactive ash revealed enlargement of the belly at the level of or just behind the pectoral fins. Livers from these animals had a yellowish brown appearance and were enlarged. Microscopic examination of some liver specimens revealed diffuse hyperplasia, hypertrophy, and fatty degeneration of hepatocytes. Ultrastructurally, there was an increased number of residual bodies containing undigested lipid, a proliferation of both rough and smooth endoplasmic reticula, and dilation of endoplasmic reticulum vesicles. The endomembrane changes correlated with observed epoxide hydrolase induction above control levels. The data obtained in these studies will be used to ascertain the safety and efficiency of incinerated low-level radioactive waste disposal in the marine environment.
The process of de novo vessel formation, called angiogenesis, is essential for tumor progression and metastasis. The identification and targeting of the molecular pathways involved in this process are becoming critical issues for anti-angiogenic cancer therapies. To pursue these molecular and pharmacological approaches, researchers need to develop better preclinical models to study tumor angiogenesis and then test anti-angiogenic therapies. As a vertebrate, the zebrafish model system is equipped with easy and powerful transgenesis and imaging tools to investigate not only angiogenesis but also tumor development and its progression. In this chapter we will illustrate how a small tropical fish can help to better understand the tumor angiogenesis process and identify new pharmacological therapies for tumor angiogenesis. Lastly, we describe in what way this model can act as a preclinical model for screening new chemical compounds which are able to selectively block tumors but not the normal healthy angiogenesis in vivo.
Protein binding of benzo(α)pyrene and mutagenicity resulting from metabolic activation by trout liver microsomal mixed function oxidase was investigated. The metabolite pattern produced by the relatively active benzo(α)pyrene hydroxylase of trout liver microsomes was established by HPLC and compared with that produced by rat liver microsomes. Three dihydrodiols, quinones and two phenols of benzo(α)pyrene were detected as well as indications of other, unidentified metabolites. The pathway of production of some of the metabolites is apparently via reactive intermediates which bind covalently to protein. Like in the mammalian system the intermediates seem to include epoxides as the inhibition of epoxide hydratase results in a relative increase in covalently bound benzo(α)pyrene. Trout liver microsomal mixed function oxidase results in formation of mutagenic intermediates from benzo(α)pyrene, aflatoxin B1 and 2-acetylaminofluorene.
Both cholangiofibroma and cholangiocarcinoma appear macroscopically as firm nodules frequently distributed in the liver in a multinodular fashion (Fig. 41). The tumor tissue usually has a grayish-white color and may also have yellow areas. The macroscopic picture may become very complex and colorful when the cholangiocellular tumors are combined with hepatocellular carcinomas or malignant mesenchymal tumors, such as angiosarcomas.
N ‐Nitroso compounds, which include nitrosamines and nitrosamides, have been known for more than 100 years, but nothing was known of their toxicological properties until 1937, when Freund described a laboratory poisoning by nitrosodimethylamine (NDMA). Then Barnes and Magee in 1954 (also following an accidental exposure of humans to NDMA being used as a solvent) described a thorough toxicological examination of the compound in several species, in which liver and/or lung injury caused death. This culminated in a chronic toxicity test in rats, which resulted in a high incidence of animals with liver tumors within a year.
The finding that a member of a large class of water‐soluble compounds was carcinogenic aroused considerable interest and an investigation began into the relationship between the chemical structure of N ‐nitroso compounds and their carcinogenic properties, initially by Druckrey et al. (mainly in rats), followed by other chemists and pathologists. The objective was to obtain clues to the mechanism(s) of carcinogenesis by these compounds, but other issues arose. One of the most interesting was the widespread nature of nitrosamine carcinogenesis that affected all species examined, although tumors of the same type were not induced in all species.
Indeed, as the number of N ‐nitroso compounds tested increased (more than 300 have been examined), it became apparent that virtually every type of human tumor was reproduced in some animal with some N ‐nitroso compound. The N ‐nitroso compounds varied widely in toxic and carcinogenic potency, but not in parallel, although the most acutely toxic compounds tended to be the most potent carcinogens. Many quite potent carcinogens, however, showed relatively low toxicity, and vice versa.
For many years, the carcinogenic N ‐nitroso compounds were considered an interesting curiosity, but in the 1960s it was found that some batches of fish meal that had been treated with sodium nitrite for preservation caused toxic liver injury in sheep. The cause of the injury was traced to nitrosodimethylamine that had formed in the fish meal. This was a surprise because nitrosamines, it was thought, formed by interaction of secondary amines with nitrite in acid solution, not at neutral pH. It has since become obvious that tertiary amines, as well as secondary amines, interact with nitrite under certain conditions (above pH 4) to form nitrosamines. Further investigations revealed that many commonly used drugs and medicines that are tertiary amines are also easily nitrosated to form N ‐nitroso compounds, thereby presenting a risk of human exposure to these carcinogens.
These old studies point out that human exposure to N ‐nitroso compounds can occur from eating nitrite‐preserved food (meat or fish) containing N ‐nitroso compounds. While deaths have occurred from acute exposure to N ‐nitroso compounds such as NDMA, epidemiological studies mostly in the 1980s and 1990s have linked childhood brain cancers with high consumption of cured meats either by their pregnant mothers or by the child. These observations are supported by animal models for several N ‐nitroso compounds documenting the induction of nervous system tumors through transplacental action of exposed pregnant animals.
Since the first report of nitrosamines (NDMA) in tobacco smoke in 1974, there has been considerable research into this topic, mainly by the group of Hoffmann and Hecht. Apart from the volatile nitrosamines, nitrosopyrrolidine, NDMA, methylnitrosoethylamine (MNEA), and nitrosodiethylamine (NDEA), a number of so‐called tobacco‐specific nitrosamines were discovered: nitrosonornicotine (NNN) and 4‐methylnitrosamino‐1‐(3‐pyridyl)‐1‐butanone (abbreviated to NNK), which is a potent carcinogen that causes liver and lung tumors in rats and hamsters and is one of the most abundant carcinogens in tobacco and tobacco smoke. NNK is also a prominent ingredient in chewing tobacco or snuff (as much as 8 ppm), one of the few carcinogens in these products (nitrosonornicotine is another, but much weaker) that certainly contributes to the carcinogenic risk to users, who not infrequently develop oral cancers. In 2007, the International Agency for Cancer Research reviewed the evidence associated with several tobacco‐specific nitrosamines and concluded the both NNN and NNK are human carcinogens (Group 1).
Other sources of exposure of humans to N ‐nitroso compounds include the air in and near factories in which nitrosamines are made or used (usually NDMA), such as those that produce or use the rocket fuel 1,1‐dimethylhydrazine, and factories in which pesticides are made, which are often stored or sold as dimethylamine salts, and which nitrogen oxides can convert to the volatile NDMA. An important source of nitrosamines is the rubber and tire industry. Nitrosamines have also been found in leather tanning establishments (mainly NDMA). Perhaps the largest industrial exposure to nitrosamines is from metalworking fluids (including cutting oils) in which concentrations of nitrosodiethanolamine (NDELA) as high as 3% have been reported, although usually less. The NDELA arises from triethanolamine (containing diethanolamine), used as an emulsifier, that combines with sodium nitrite used as a corrosion inhibitor. A combination of an alkanolamine, an aldehyde, and a nitrite, as may be present in a metalworking fluid, can also give rise to cyclic nitrosamines containing oxygen, such as a nitrosooxazolidine, that are potent carcinogens. The Consumer Products Commission and the Federal Drug Administration have reported that nitrosamines have been found in pacifiers and baby bottle nipples and can migrate into saliva. NDMA is receiving significant attention over the past decade because it can be present in drinking water as a by‐product of water chlorination from treatment plants that use chloramines for disinfection. It can also be found in ground water at levels as high as 10 ppm as NDMA is formed by the reaction of commonly occurring primary, secondary, or tertiary amines with nitrates.
Arylamines are chemically synthesized and contained in oxidants, epoxy polymers, explosives, fungicides, pesticides, colorants, polyurethanes, and used in rubber, pharmacology, cosmetics, and other chemical industries. Many arylamines are ubiquitously present in cigarette smoke, cooking fume hoods, foods, automobile exhaust, industrial sites, etc. Some arylamines can be generated through azo reduction by intestinal, skin, and environmental microorganisms from azo dyes that are widely used. Arylamines can also be generated by reduction of the nitro-group containing polyhydrated hydrocarbons including muntions. Some arylamines are released by burning nitrogen containing organic materials at high temperatures. Some medical drugs are also arylamines. Furthermore, many arylamines are essential constituents of normal metabolism or the result of abnormal metabolism or dietary sources. Some arylamines are mutagenic, carcinogenic or the cause of other kinds of maladies. Some arylamine are considered the major etiological agents of bladder tumors in humans and animals but may also induce other types of cancers in various organs. The organ, tissue, and species specificity of the arylamine-inducing carcinogenesis may be determined by their availability, distribution, and the presence of metabolic activation/detoxicification enzymes of each organ or tissue of different species. The ubiquitous arylamines, therefore, pose serious hazards to human health and environment. This article will address the occurrence, uses/application, and carcinogenicity of some arylamines. Research perspectives on the the arylamines that induce other diseases will also be discussed.
Bullhead catfish, Ictalurus nebulosus, and rainbow trout, Salmo gairdneri, were subjected to two coal-derived substances to evaluate each material for potential carcinogenic activity. Substances tested included a solvent-refined coal heavy distillate and a sediment extract from an industrially polluted river in Ohio. Two subfractions, one containing the polycyclic aromatic hydrocarbons and the other containing the nitrogen heterocycles of each material were also tested. Following a 16-h exposure period, the gill and liver tissues were removed and examined by standard light and electron microscopy techniques. Hyperplasia of the gill tissue and some engorgement with blood of the secondary lamellae were observed with the light microscope. At the electron microscope level, hepatocyte mitochondria were swollen with a loss of cristae, and rough endoplasmic reticulum fragmented. Results suggest that most of the treatments studied induced pathological responses in fish. Of the two fish tested, the rainbow trout was the most sensitive indicator of environmental pollution.
Diethyl- and dimethylnitrosamines, dissolved in aquarium water in doses of 200–400 ppm, induced the formation of neoplasms of the digestive gland (basophil-cell tumors) in the molluskUnio pictorum in 16 of 95 and 6 of 17 animals, respectively, which survived until the time of appearance of the first tumor (38–39 days), and also neoplasms of the hematopoietic system (lymphatic leukemia) in seven and one mollusks, respectively, mainly in conjunction with tumors of the digestive gland. Metylnitronitrosoguanidine caused only inflammatory changes at the site of the injection. The mollusks can be used with advantage as a biological indicator of pollution of the hydrosphere with chemical carcinogens.
Development of carcinogenesis bioassays that utilize small fish species is important principally for investigating the causes of neoplasms in wild fishes and for providing alternative or supplementary models to rodent carcinogenicity tests. Availability, economy, latency of tumorigenic response, and ease of maintenance and exposure are commonly cited advantages of small fish species in carcinogenesis bioassays. Carcinogen metabolism and mechanisms of carcinogenesis in several small fish species appear similar to those processes in the more thoroughly studied large fish species as well as in rodents. Recent studies suggest that small fish species are appropriate test models for waterborne carcinogens having a variety of mechanisms. Several small fish species readily develop hepatic and non-hepatic neoplasms following brief static exposures to direct-acting genotoxic compounds such as methylazoxymethanol acetate (MAM-Ac) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Tumorigenic responses appear to be related to species and to various exposure factors. Indirect-acting genotoxic carcinogens such as nitrosamines and polynuclear aromatic hydrocarbons have not been thoroughly tested in small fish species but appear to require longer exposures than direct-acting ones to induce hepatic neoplasms. An especially important potential use of small fish carcinogenesis bioassays is in testing carcinogens that might have epigenetic mechanisms, especially complex mixtures that might affect man or the environment but are difficult to test in rodent models. Preliminary studies indicate that prolonged exposures of up to 6 months to a mixture of halogenated organic compounds result in hepatic neoplasms in two small fish species. To improve their usefulness and exploit small fish carcinogenesis models better, gaps in several areas need to be filled. These include (1) a better understanding of biological and nutritional requirements of test species as related to carcinogenesis, (2) a broader database on neoplastic responses of various species to various chemicals, and (3) development of special exposure methods and standardization of test protocols.
The feasibility of using fish tumours as indicators for chemical carcinogens in surface waters in The Netherlands was studied. Fish from surface waters with a different degree of chemical pollution and mutagenic activity were examined.Amongst 4184 fish from the river Rhine and its branches, 5 bream (Abramis brama) showed liver neoplasma (hepatoma, hepatocellular carcinoma) and 2 roach (Rutilus rutilus) exhibited thyroid tumours. Of 1435 fish from the river Meuse 1 bream showed a hepatocellular carcinoma. No tumours were found in 714 fish from Lake IJssel, nor in 1404 fish from the relatively clean Lake Braassem. Although it is likely that chemical carcinogens are responsible for the induction of at least the liver tumours, the paucity of positive findings in fish from the polluted river Rhine areas is a warning against the use of feral fish as bioindicators for environmental chemical carcinogens.
Phosphotyrosyl protein phosphatase was purified from the liver of the Tilapia mossambica. Specific activity of the final purified preparation was 5,095 U/mg of protein with the 32P-peptide 1142–1153 of the insulin receptor as substrate. SDS-polyacrylamide gel electrophoresis revealed that monomers of the phosphotyrosyl protein phosphatase have relative masses of 67,000 ± 500 and 60,000 ± 500. Since the active phosphotyrosyl protein phosphatase was found to have a relative mass of 62,000 ± 1,000, the purified enzyme was concluded to be monomeric. The phosphotyrosyl protein phosphatase had an isoelectric point of 5.79 ± 0.02 and an optimal pH of 7.0. Incubation of the purified enzyme fraction with Src-phosphorylated DNA topoisomerase I resulted in an increase activity of DNA topoisomerase I in a dose-dependent manner. Activity of phosphotyrosyl protein phosphatase is inhibited by MnCl2 and vanadate at μmolar concentrations, but less inhibition was observed with CaCl2 and MgCl2. J. Exp. Zool. 277:14–22, 1997. © 1997 Wiley-Liss, Inc.
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.
Neoplastic diseases, particularly of bottom-dwelling fishes, are more prevalent in coastal areas than in areas that are relatively pristine. Although sediments in many urbanized estuaries contain high concentrations of contaminants, there is little evidence linking a specific organic or inorganic chemical to a particular liver lesion in winter flounder (Pleuronectes americanus), despite increasing study in recent years. Between 1984 and 1986, sediments and winter flounder were collected from 10 sites in the north-east United States ranging from grossly polluted to relatively unimpacted. Sediments were analysed for polycyclic aromatic hydrocarbons (PAHs), chlorinated pesticides, polychlorinated biphenyls (PCBs), and metals. Gross and microscopic pathological examinations were conducted on winter flounder liver sections. Factor and canonical correlation analyses were used to explore associations between biological and chemical measurements. Hepatitis, cholangitis, phlebitis, and macrophage aggregate hyperplasia showed positive associations with low molecular weight, petroleum-derived PAHs (naphthalene, anthracene, 1-methylnaphthalene, 2-methylnaphthalene, 2,6-dimethylnaphthalene, acenaphthene, phenanthrene, biphenyl, fluorene, and 1-methylphenanthrene), the pesticides lindane, hexachlorobenzene, heptachlor epoxide, and o,p'-DDD, tri- to hexachlorobiphenyls, and chromium, cadmium, lead, thallium, and selenium, but were negatively associated with the pesticides o,p'-DDT, p,p'-DDT, p,p'-DDE, and mirex. Cytoplasmic hepatocellular vacuolation, cytoplasmic bile duct vacuolation, neoplasms, and pre-neoplasms showed positive associations with most PAHs measured, whether petroleum-derived or combustion-derived (benzo[a]pyrene, benzo[e]pyrene, benzo[a]anthracene, dibenzo[a,h]anthracene, perylene, chrysene, and fluoranthene), the pesticides dieldrin, trans-nonachlor and alpha-chlordane, and silver, copper, antimony, and tin, but no associations with PCBs were found. Coagulative necrosis, single cell necrosis and haemorrhagic necrosis showed positive associations with hepta- to nona-chlorobiphenyls and arsenic, zinc, nickel, and mercury, and negative associations with high molecular weight, combustion-derived PAHs and DDT compounds and metabolites.
Epizootiological analyses of field data on prevalances of hepatic neoplasms and other hepatic lesions in English sole (Parophrys vetulus) from Puget Sound suggest a link between these lesions and sediment-associated xenobiotics, particularly aromatic hydrocarbons (AHs). To further investigate this relationship, English sole from a minimally contaminated site were parenterally exposed at monthly intervals for 1 yr to an organic solvent extract of a contaminated urban sediment containing high levels of AHs (30 or 75 mg/kg body weight) or to the model carcinogen benzo[a]pyrene (BaP, 12 mg/kg body weight). Control groups included English sole exposed to an extract of a relatively uncontaminated reference sediment, sole exposed to the carrier only, and untreated sole. Eighteen months after the initial injections, examination of livers revealed a significantly different (increased, ) incidence of a spectrum of hepatic lesions, including presumptive preneoplastic foci of cellular alteration, in sole exposed to the urban sediment extract or BaP. Sole in control groups did not develop any of these hepatic lesions. These results represent the first direct demonstration of the hepatotoxicity of sediment-associated chemical contaminants in English sole and strengthen the case for a cause-and-effect relationship between exposure to AHs and epizootic neoplastic hepatic lesions in this species.
Explant and monolayer cell cultures were initiated from oyster heart and embryonic tissue and maintained for periods of a few days to 6 mo depending on the type of tissue and the culture medium. A pH of 7.0–7.3 and a temperature of 20 °C were optimum. Vertebrate cell culture media prepared in a marine saline and supplemented with fetal bovine serum and protein digests provided a suitable basal medium. Supplementation of the basal medium with oyster hemolymph or extracts of oyster tissue markedly prolonged cell maintenance. Explant cultures of heart tissue with the subsequent outward migration of individual cells were most easily initiated and maintained for periods up to 6 mo. Although several cell types were observed, actively motile, granular amoebocytes predominated. No mitotic cells were observed even following exposure to a variety of mitogens. Cultures initiated from disaggregated larvae did yield actively dividing cells. Key words: oyster, cell culture, amoebocytes
A relatively simple and reliable in vitro method for marine fish chromosome study was developed. The addition of 10% chick embryo extract to serum-supplemented Eagle's minimum essential medium with high concentration of NaCl resulted in marked growth of kidney cells in the marine conger eel (Astroconger myriaster) after activation by phytohemagglutinin (PHA). Culture medium without chick embryo extract or PHA and/or with normal concentration of NaCl did not induce substantial growth. In contrast to reports by others, humidified culture was not required for excellent cell growth of these teleost kidney cells. Numerous metaphases unmarred by overlapping chromosomes were recovered and excellent karyograms were available for detailed karyotype analysis. Key words: kidney, culture, marine fish, chromosome
During the past decade, the zebrafish has emerged as a leading model for mechanistic cancer research because of its sophisticated genetic and genomic resources, its tractability for tissue targeting of transgene expression, its efficiency for forward genetic approaches to cancer model development, and its cost effectiveness for enhancer and suppressor screens once a cancer model is established. However, in contrast with other laboratory animal species widely used as cancer models, much basic cancer biology information is lacking in zebrafish. As yet, data are not published regarding dietary influences on neoplasm incidences in zebrafish. Little information is available regarding spontaneous tumor incidences or histologic types in wild-type lines of zebrafish. So far, a comprehensive database documenting the full spectrum of neoplasia in various organ systems and tissues is not available for zebrafish as it is for other intensely studied laboratory animal species. This article confirms that, as in other species, diet and husbandry can profoundly influence tumor incidences and histologic spectra in zebrafish. We show that in many laboratory colonies wild-type lines of zebrafish exhibit elevated neoplasm incidences and neoplasm-associated lesions such as heptocyte megalocytosis. We present experimental evidence showing that certain diet and water management regimens can result in high incidences of neoplasia and neoplasm-associated lesions. We document the wide array of benign and malignant neoplasms affecting nearly every organ, tissue, and cell type in zebrafish, in some cases as a spontaneous aging change, and in other cases due to carcinogen treatment or genetic manipulation.
Zebrafish (Brachydanio rerio) were evaluated as a small fish model for environmental carcinogenesis monitoring. Criteria used for evaluation included: (1) sensitivity to dose response exposures of 6 known carcinogens by 4 routes of exposure, (2) histopathologic evaluation of the resulting lesions and comparison with responses in other species, (3) response to promoters of neoplasia, (4) ability to conduct carcinogen metabolism studies to understand mechanisms of action, (5) determining the role of oncogenes in zebrafish carcinogenesis, and (6) success in developing transgenics that would increase the species' usefulness as a carcinogen monitoring model. Findings were (1) zebrafish did respond to known carcinogens with variable but overall acceptable sensitivity, (2) the histopathology of their responses was similar to that observed in other small fish species, (3) they did not respond well in limited promotional studies, (4) metabolism studies were very difficult due to small fish and organ size and results did not always support observed tumor responses, (5) ras and p53 genes were sequenced but lack of grossly observable tumors prevented the determination of mutations in these genes, and (6) although progress was made, the production of transgenic fish was not achieved. Although none of these results would disqualify zebrafish as environmental monitors, their strict requirement for tropical or subtropical water temperatures would make them completely unsuitable for temperate water use.
Contaminants in water and sediments can be carcinogenic to aquatic wildlife as well as humans. Identifying those carcinogens, however, is difficult because they often occur in low concentrations and exert their effects over a large part of the life span of affected organisms. Furthermore, the carcinogens are often components of complex mixtures. Recent studies suggest that laboratory-reared fish species might be well suited for testing water-associated and other carcinogens. Here, we review the principal carcinogen exposure methods that utilize small fish species or can be adapted to utilize small fish species to detect carcinogens in aqueous environments. Emphasis is placed on methods for which the end-point is tumor induction. The methods discussed are dietary exposures, skin painting, embryo microinjection, early life stage (pulse) exposures, static water exposures, flow-through exposures, and controlled field exposures. Early life stage exposures seem to have the greatest utility with regard to carcinogen sensitivity, ease of administration, disposal of test compounds, and economy of materials and effort. For certain types of carcinogens, however, long-term flow-through exposures are probably required. In summary, small fish carcinogenesis models offer an array of methodologies that can be utilized in a variety of combinations depending on compounds tested, exposure parameters employed, and end points sought.
N ‐Nitroso compounds, which include nitrosamines and nitrosamides, have been known for more than 100 years, but nothing was known of their toxicologic properties until 1937, when Freund described a laboratory poisoning by nitrosodimethylamine (NDMA). Then Barnes and Magee in 1954 (also following an accidental exposure of humans to NDMA being used as a solvent) described a thorough toxicological examination of the compound in several species, in which liver and/or lung injury caused death. This culminated in a chronic toxicity test in rats, which resulted in a high incidence of animals with liver tumors within a year.
The finding that a member of a large class of water‐soluble compounds was carcinogenic aroused considerable interest and an investigation began into the relationship between the chemical structure of N ‐nitroso compounds and their carcinogenic properties, initially by Druckrey et al. (mainly in rats), followed by other chemists and pathologists. The objective was to obtain clues to the mechanism(s) of carcinogenesis by these compounds, but other issues arose. One of the most interesting was the widespread nature of nitrosamine carcinogenesis that affected all species examined, although not always were tumors of the same type induced in all species.
Indeed, as the number of N ‐nitroso compounds tested increased (more than 300 have been examined), it became apparent that virtually every type of human tumor was reproduced in some animal with some N ‐nitroso compound. The N ‐nitroso compounds varied widely in toxic and carcinogenic potency, but not in parallel, although the most acutely toxic compounds tended to be the most potent carcinogens. Many quite potent carcinogens, however, showed relatively low toxicity, and vice versa.
For many years, the carcinogenic N ‐nitroso compounds were considered an interesting curiosity, but in the 1960s it was found that some batches of fish meal which had been treated with sodium nitrite for preservation caused toxic liver injury in sheep. The cause of the injury was traced to nitrosodimethylamine (NDMA) which had formed in the fish meal. This was a surprise because nitrosamines, it was thought, formed by interaction of secondary amines with nitrite in acid solution, not at neutral pH. It has since become obvious that tertiary amines, as well as secondary amines, interact with nitrite under certain conditions (above pH 4) to form nitrosamines. This was information previously known but, like Freund's report, was buried in the literature. Further investigations revealed that many commonly used drugs and medicines which are tertiary amines are also easily nitrosated to form N ‐nitroso compounds, thereby presenting a risk of human exposure to these carcinogens. In the case of the nitrite‐treated fish meal, it is not clearly known whether the NDMA arises by nitrosation of dimethylamine, trimethylamine, trimethylamine‐ N ‐oxide, or some other precursor. In addition to nitrites, nitrosation can also be effected by “nitrous gases” (nitrogen oxides) in burning fuel, by alkyl nitrites, or by (often biologically inactive) nitrosamines, such as nitrosamino acids, through a process called transnitrosation.
These old studies point out that human exposure to N ‐nitroso compounds can occur from eating nitrite‐preserved food (meat or fish) containing N ‐nitroso compounds. There was, and possibly still is, NDMA in beer, which arises from the interaction of alkaloids (hordenine and gramine) and other tertiary amines in the malt with nitrogen oxides in the gases used to heat the malt; up to 50 parts per million of NDMA has been reported in certain beers. The search for alkylnitrosoureas in cured meats is prompted by some epidemiological observations that linked brain cancers of children with high consumption of cured meats by their pregnant mothers and the fact that probably the best animal model for inducing tumors of the nervous system is the transplacental action of alkylnitrosoureas in pregnant rats or mice.
Since the first report of nitrosamines (NDMA) in tobacco smoke in 1974, there has been considerable research into this topic, mainly by the group of Hoffmann and Hecht. Apart from the volatile nitrosamines, nitrosopyrrolidine, NDMA, NMEA, and NDEA, a number of so‐called “tobacco‐specific” nitrosamines were discovered: nitrosonornicotine and 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (abbreviated to NNK), which is a potent carcinogen that causes liver and lung tumors in rats and hamsters and is one of the most abundant carcinogens in tobacco and tobacco smoke. NNK is also a prominent ingredient in chewing tobacco or snuff (as much as 8 ppm), one of the few carcinogens in these products (nitrosonornicotine is another, but much weaker) which certainly contributes to the carcinogenic risk to users, who not infrequently develop oral cancers.
Other sources of exposure of humans to N ‐nitroso compounds include the air in and near factories in which nitrosamines are made or used (usually NDMA), such as those that produce or use the rocket fuel 1,1‐dimethylhydrazine, and factories in which pesticides are made, which are often stored or sold as dimethylamine salts, and which nitrogen oxides can convert to the volatile NDMA. An important source of nitrosamines is the rubber and tire industry. Nitrosamines have also been found in leather tanning establishments (mainly NDMA). Perhaps the largest industrial exposure to nitrosamines is from metalworking fluids (including cutting oils) in which concentrations of nitrosodiethanolamine (NDELA) as high as 3% have been reported, although usually less. The NDELA arises from triethanolamine (containing diethanolamine), used as an emulsifier, that combines with sodium nitrite used as a corrosion inhibitor. A combination of an alkanolamine, an aldehyde, and a nitrite, as may be present in a metalworking fluid, can also give rise to cyclic nitrosamines containing oxygen, such as a nitrosooxazolidine, that are potent carcinogens. The common use of nitrites as corrosion inhibitors for cans leads to contamination of many amines that are shipped in cans and is responsible for the presence of nitrosamines such as methylnitrosododecylamine and methylnitrosotetradecylamine in shampoos and other personal hygiene preparations. Nitrosamines have been reported in soil, water, and in sewage, but information is incomplete.
Much of the parenchyma of the normal pronephros of the adult Fundulus heterodilus, a euryhaline teleost, is haematopoietic tissue which was examined in cytocentrifuge preparations and plastic embedded thick sections. As we have not characterized many of the blood cells functionally, the terminology used is based on their morphological resemblance to similarly named cells in higher vertebrates. Approximately 80% of the non-erythroid elements observed in the pronephros are mature eosinophilic granulocytes (48%), immature eosinophilic granulocytes (25%), or cells likely to be their precursors [e.g. small blast cells (6%) and large blast cells (2%)]. Although there are macrophages in the pronephros that are capable of endocytotic activity, the mature and immature granulocytes are not. The granulocytes are non-specific esterase positive, PAS positive, acid phosphatase negative, and are capable of being mobilized by a RES activating agent, Ecteinascidia turbinata.
The registry is mainly involved in specimen storage, diagnostics, information retrieval, and other service activities. In addition, however, the registry participates in surveys, identifies unique lesions or tumorous populations, and takes a supportive or dominant role in pertinent research projects. It is hoped that these efforts will benefit the fight against cancer by contributing further to the discovery of new carcinogens, indicators of environmental carcinogens, animal models for cancer studies, efficacious methods for screening carcinogens, and of reservoirs of carcinogens in food sources and by helping to provide an overall understanding of the neoplastic process.
Guppy and medaka developed hepatocellular neoplastic lesions after water-borne exposure to the model procarcinogen 2-acetylaminofluorene (AAF), although with the numbers studied the incidence of tumors was statistically significant only in guppies. A major pathway for bioactivation of AAF is N-hydroxylation followed by sulfation. The metabolism of [14C]-AAF was studied with liver microsomes from pools of control or AAF-exposed (1 mg/L for 4 days) guppy and medaka. Sulfotransferase (ST) and glucuronyltransferase (UGT) activities were respectively measured in cytosol or detergent-treated microsomes with 4-methylumbelliferone and N-, 3- or 7-hydroxyAAF as substrates. Total AAF monooxygenation was more rapid in untreated guppies (0.52±0.03 nmole min−1 mg−1 protein) than medaka (0.26 nmole min−1mg−1 protein). AAF monooxygenation was reduced in the acutely AAF-exposed fish. The major monooxygenated metabolites formed by all microsomes were 7-hydroxyAAF (80% of total) and 5-hydroxyAAF (10%). Only small amounts of N-hydroxyAAF were detected, consistent with the low carcinogenicity. AAF-treated guppies had higher ST activity than controls, but UGT activity was reduced or unaffected by AAF exposure. No increase in ST was observed in medaka, but UGT activity was slightly increased by AAF exposure. These different effects of AAF exposure on conjugating enzyme activities in guppies and medaka may partially explain the greater carcinogenic sensitivity of guppies to AAF.
Rainbow trout (Salmo gairdneri) embryos were exposed in ovo to benzo[a]pyrene (BaP) by a single transchorionic injection. Injected BaP was retained in the yolk sac of larval trout and was only slowly metabolized until swim-up. One year post-exposure, BaP-treated fish had an elevated incidence of liver neoplasms which ranged from small basophilic microfoci of altered hepatocytes to large hepatocellular carcinomas. These results demonstrate carcinogenesis in rainbow trout from single dose exposure to BaP, a widely distributed environmental pollutant that has been implicated as the cause of liver neoplasia in wild fish and some human cancers.
Chronic exposure to arylamines through diet and/or smoking has been associated with genetic changes and tumorigenesis. Cellular proliferation, apoptosis, and histological changes in liver tissue were investigated in Gambusia affinis (G affinis) after chronic dietary exposure to 6.9 mM and 0.069 mM concentrations of benzidine (BZ), 2-aminofluorene (2AF), and their combination for 4, 8, and 12 weeks, respectively. The proliferation assay indicated non-dose-dependent increases in cellular proliferation over the controls for all treatment groups at 4 and 12 weeks but not at 8 weeks except for the low dose of 2AF. The apoptosis assay showed effects in the low-dose group of 2AF and BZ at 4 weeks only. Hematoxylin/eosin staining of liver tissue revealed an increase in oval/spindle cell proliferation and altered foci formation in the treated groups compared with controls. These results demonstrate a mammalian-like response to 2AF and BZ in G affinis liver.
Metabolic activation and covalent binding of benzo[a]pyrene (BP) to deproteinized salmon sperm DNA by supernatant fractions (10,000 g) of liver homogenates isolated from untreated, 3-methylcholanthrene (3-MC) or BP-treated starry flounder (Platichthys stellatus) and coho salmon (Oncorhynchus kisutch) were investigated. The influence of temperature, pH, time and concentrations of protein, BP, NADPH and DNA on covalent binding was investigated to obtain optimum conditions for in vitro binding (pmoles of BP equivalents bound/mg DNA/mg protein) of [(3)H]BP to DNA for each of the two fish species. When the supernatant fractions from untreated starry flounder were used, the covalent binding of BP to DNA was 7.5 and 2.5 times greater than the values obtained with the supernatant fractions from untreated coho salmon or rat respectively. Treatment of both fish species with 3-MC or BP resulted in a marked (10- to 53-fold) increase in the binding. Ethyl acetate-extractable metabolites formed by fish liver supernatant fractions consisted of BP dihydrodiols (4,5-, 7,8-, and 9,10-dihydrodiols), phenols (3-OH, 9-OH, and 7-OH), quinones (3,6-, 1,6- and 6,12-Q) and BP 4,5-oxide. For both fish species, BP 9,10-dihydrodiol and BP 7,8-dihydrodiol were the major metabolites comprising as much as 48-72 per cent of the total ethyl acetate-extractable metabolites; 3-hydroxy BP was also present in significant amounts. The ratio of the non-K region dihydrodiols to phenols was significantly greater for both fish species compared to rat.
About 7000 animals of 65 different genotypes of the xiphophorine fish were treated with the direct acting chemical carcinogen N-methyl-N-nitrosourea (MNU; 10(-3)M; four times for 1 hour in two week intervals), in order to find out whether the susceptibility for development of fibrosarcomas and rhabdomyosarcomas is directly related to the genotype. A genotype specific susceptibility was found, ranging from zero to about nine percent. The highest susceptibles were found in certain backcross hybrids involving P.variatus/X.helleri-hybrids and X.helleri, as the recurrent parent. These genotypes were further analysed. Both P.variatus and X.helleri, as werr as their F1 proved to be insusceptible; while from the three backcrosses, which were tested, namely the BC1, BC4 and BC15, both the BC1, and the BC4, were susceptible, but the BC15 was insusceptible. The results are interpreted on the basis of the assumption that the differential susceptibility is a function of the type of control of a tumor gene (Tu-Fi-Rh) endogenous to P.variatus and involved in development of fibrosarcomas and rhabdomyosarcomas. Accordingly, in P.variatus and in the F1 the Tu-Fi-Rh is controlled by repressing genes (R-genes) linked as well as non-linked to Tu-Fi-Rh; because simultaneous mutation of both R-genes following treatment with MNU is an extremely unlikely event, these genotypes have an extremely low susceptibility. By contrast, in the BC1 and the BC4 the non-linked R-genes become eliminated and only the linked R-gene remains for repression of Tu-Fi-Rh; this condition confers a high degree of susceptibility, because one single mutation may lead to impairment of the R-gene and to Tu-Fi-Rh-mediated formulation of fibrosarcomas and rhabdomysarcomas. In the BC15, furthermore, also the Tu-Fi-Rh has become eliminated, resulting in a loss of the susceptibility. The results suggest that in the xiphophophorine fish the susceptibility for responding to MNU-treatment with the development of fibrosarcomas and rhabdomysarcomas is related directly to the genotype.
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