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Fish Models for Environmental Carcinogenesis: The Rainbow Trout
Abstract
Progress over the past 30 years has revealed many strengths of the rainbow trout as an alternative model for environmental carcinogenesis research. These include low rearing costs, an early life-stage ultrasensitive bioassay, sensitivity to many classes of carcinogen, a well-described tumor pathology, responsiveness to tumor promoters and inhibitors, and a mechanistically informative nonmammalian comparative status. Low-cost husbandry, for example, has permitted statistically challenging tumor study designs with up to 10,000 trout to investigate the quantitative interrelationships among carcinogen dose, anticarcinogen dose, DNA adduct formation, and final tumor outcome. The basic elements of the trout carcinogen bioassay include multiple exposure routes, carcinogen response, husbandry requirements, and pathology. The principal known neoplasms occur in liver (mixed hepatocellular/cholangiocellular adenoma and carcinoma, hepatocellular carcinoma), kidney (nephroblastoma), swim bladder (adenopapilloma), and stomach (adenopapilloma). Trout possess a complex but incompletely characterized array of cytochromes P450, transferases, and other enzymic systems for phase I and phase II procarcinogen metabolism. In general, trout exhibit only limited capacity for DNA repair, especially for removal of bulky DNA adducts. This factor, together with a high capacity for P450 bioactivation and negligible glutathione transferase-mediated detoxication of the epoxide, accounts for the exceptional sensitivity of trout to aflatoxin B1 carcinogenesis. At the gene level, all trout tumors except nephroblastoma exhibit variable and often high incidences of oncogenic Ki-ras gene mutations. Mutations in the trout p53 tumor suppressor gene have yet to be described. There are many aspects of the trout model, especially the lack of complete organ homology, that limit its application as a surrogate for human cancer research. Within these limitations, however, it is apparent that trout and other fish models can serve as highly useful adjuncts to conventional rodent models in the study of environmental carcinogenesis and its modulation. For some problems, fish models can provide wholly unique approaches.
... Based on these features, these tumors were diagnosed as adenomas originating from the gas glandular epithelium of the swim bladder. Additionally, these tumor cells did not seem to function as gas glandular epithelium, since the swim bladder lumen did not form in Swim bladder tumors can be induced in teleosts via exposure to environmental contaminants and carcinogens 17,18 . The chemical-induced swim bladder tumors are reported in the medaka exposed to 4-chloroaniline 19 , aniline 19 , N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 20 , or bis (tri-n-butyltin)oxide 21 ; in the guppy exposed to methyl mercury chloride 22 ; and in the rainbow trout exposed to diethylnitrosamine 17 17 , or 2,6-dimethylnltrosomorpholine 23 . ...
... Additionally, these tumor cells did not seem to function as gas glandular epithelium, since the swim bladder lumen did not form in Swim bladder tumors can be induced in teleosts via exposure to environmental contaminants and carcinogens 17,18 . The chemical-induced swim bladder tumors are reported in the medaka exposed to 4-chloroaniline 19 , aniline 19 , N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 20 , or bis (tri-n-butyltin)oxide 21 ; in the guppy exposed to methyl mercury chloride 22 ; and in the rainbow trout exposed to diethylnitrosamine 17 17 , or 2,6-dimethylnltrosomorpholine 23 . Conversely, spontaneous swim bladder tumors are rare in teleosts, with an incidence of 0.02% (2/10,000) in medakas than 24 weeks of age and 0.14% (7/5,000) in guppies older than 13 weeks of age, which have been used in the control groups of a variety of carcinogenesis tests 11 . ...
... Additionally, these tumor cells did not seem to function as gas glandular epithelium, since the swim bladder lumen did not form in Swim bladder tumors can be induced in teleosts via exposure to environmental contaminants and carcinogens 17,18 . The chemical-induced swim bladder tumors are reported in the medaka exposed to 4-chloroaniline 19 , aniline 19 , N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 20 , or bis (tri-n-butyltin)oxide 21 ; in the guppy exposed to methyl mercury chloride 22 ; and in the rainbow trout exposed to diethylnitrosamine 17 17 , or 2,6-dimethylnltrosomorpholine 23 . Conversely, spontaneous swim bladder tumors are rare in teleosts, with an incidence of 0.02% (2/10,000) in medakas than 24 weeks of age and 0.14% (7/5,000) in guppies older than 13 weeks of age, which have been used in the control groups of a variety of carcinogenesis tests 11 . ...
Swim bladder tumors were detected in three out of 28 wavy medakas aged about 2 years old, all of which displayed abnormal swimming patterns caused by their spinal curvature. The tumors were located in the dorsal abdominal cavity. The swim bladder lumen was not detected in the region where it was originally assumed to be located, and that region was replaced with adipose tissue. The tumors were non-invasive, expansile, and encapsulated solid masses composed of a homogenous population of well-differentiated, densely packed, gas glandular epithelium-like cells. The tumor masses were connected to the rete mirabile, but the tumor cells did not infiltrate into them. Histopathologically, these tumors were diagnosed as adenomas originating from the gas glandular epithelium of the swim bladder. Spontaneous swim bladder tumors are rare in medaka, with an incidence of 0.02%; however, in the present study of wavy medaka, the incidence was much higher (10.7%). The long-term physical effects on the gas gland caused by swim bladder deformation considered to be a secondary effect of the spinal curvature may be an important factor in the proliferation of the gas glandular epithelium in the wavy medaka, resulting in the higher incidence of swim bladder tumors.
... 18 Studies using these fish have revealed a low propensity for spontaneous tumors; rather, they require exposure to carcinogens and mutagens to induce tumor formation. 3,12,[19][20][21] Information concerning the etiologies of neoplasms in wild and production fishes may, therefore, provide information applicable or relevant to ornamental and hobby fish seen by the veterinary practitioner. For example, up to 10% of salmon (Salmo salar) from a commercial aquaculture facility fed commercial pellets without any known potential carcinogens in the feed developed intestinal adenocarcinomas and other types of tumors. ...
... Aggressive carcinomas and mixed malignant neoplasms of the intestine in the zebrafish, however, have been associated with the nematode Pseudocapillaria tomentosa. 80 Gastric adenopapillomas have been reported in rainbow trout (Oncorhynchus mykiss) exposed to chemicals, 20 whereas esophageal and gastric adenomas and invasive adenomas and adenocarcinomas of the intestine have also been induced in laboratory fish exposed to chemical agents. 3 Hepatobiliary neoplasms are the most common visceral neoplasms of fishes, although an accurate determination of the prevalence of these neoplasms is difficult to establish in nonlaboratory fishes, because internal neoplasms are obviously less apparent than external neoplasms. ...
... 3 For example, aflatoxins have been shown to induce hepatobiliary neoplasms in rainbow trout, which is particularly susceptible to this toxin among vertebrates. 19,20 Cottonseed meal incorporated in pelleted diets can become infected by toxin-producing Aspergillus spp and the cyclopropenoid fatty acids of cottonseeds enhance the carcinogenic effect of aflatoxins. 19 Feeds that contain an increased amount of lipids associated have been shown to result in steatohepatitis and hepatocellular carcinomas in medaka exposed to carcinogenic agents. ...
The scientific literature contains a wealth of information concerning spontaneous fish neoplasms, although ornamental fish oncology is still in its infancy. The occurrence of fish neoplasms has often been associated with oncogenic viruses and environmental insults, making them useful markers for environmental contaminants. The use of fish, including zebrafish, as models of human carcinogenesis has been developed and knowledge gained from these models may also be applied to ornamental fish, although more studies are required. This review summarizes information available about fish oncology pertaining to veterinary clinicians.
... 18 Studies using these fish have revealed a low propensity for spontaneous tumors; rather, they require exposure to carcinogens and mutagens to induce tumor formation. 3,12,[19][20][21] Information concerning the etiologies of neoplasms in wild and production fishes may, therefore, provide information applicable or relevant to ornamental and hobby fish seen by the veterinary practitioner. For example, up to 10% of salmon (Salmo salar) from a commercial aquaculture facility fed commercial pellets without any known potential carcinogens in the feed developed intestinal adenocarcinomas and other types of tumors. ...
... Aggressive carcinomas and mixed malignant neoplasms of the intestine in the zebrafish, however, have been associated with the nematode Pseudocapillaria tomentosa. 80 Gastric adenopapillomas have been reported in rainbow trout (Oncorhynchus mykiss) exposed to chemicals, 20 whereas esophageal and gastric adenomas and invasive adenomas and adenocarcinomas of the intestine have also been induced in laboratory fish exposed to chemical agents. 3 Hepatobiliary neoplasms are the most common visceral neoplasms of fishes, although an accurate determination of the prevalence of these neoplasms is difficult to establish in nonlaboratory fishes, because internal neoplasms are obviously less apparent than external neoplasms. ...
... 3 For example, aflatoxins have been shown to induce hepatobiliary neoplasms in rainbow trout, which is particularly susceptible to this toxin among vertebrates. 19,20 Cottonseed meal incorporated in pelleted diets can become infected by toxin-producing Aspergillus spp and the cyclopropenoid fatty acids of cottonseeds enhance the carcinogenic effect of aflatoxins. 19 Feeds that contain an increased amount of lipids associated have been shown to result in steatohepatitis and hepatocellular carcinomas in medaka exposed to carcinogenic agents. ...
... Microinjection is a micromanipulation technique invented in 1902 by Dr. Marshall Barber that is currently instrumental in biomedical research (from transgenics, gene targeting, animal cloning, human infertility treatment, and nuclease-guided genetic engineering to RNA-guided genome editing) and healthcare. In aquaculture, microinjections are used in genetic, molecular, and toxicological research (Metcalfe and Sonstegard 1984, Black et al. 1985, Metcalfe et al. 1988, Inoue 1992, Bailey et al. 1996, Okhyun et al. 2015, Goto et al. 2019, Michiels et al. 2019. Microinjection is a very precise technique, which permits inserting/retrieving substances at a microscopic or macroscopic level into/out of a single living organelle, cell, or multicellular entity using a needle, usually a glass micropipette 0.5 to 15 μm in diameter. ...
... One of the main advantages of microinjection is the wide variety of substances that can be delivered, such as DNA, RNA, protein, and other macromolecules (Elaswad et al. 2018, Goto et al. 2019, Xu 2019. Salmonid microinjections described in the literature have many applications: genetic manipulation (mainly production of transgenic fish), in vitro fertilization, cryopreservation technology, and toxicological tests (Bailey et al. 1996, Shirakashi et al. 2015. Moreover, thanks to the possibility of administering precise doses, the microinjection method can also be used to administer other substances, including carcinogens (Black et al. 1985) or infectious agents, into eggs and embryos, to simulate the vertical transmission of pathogens. ...
The paper describes an experimental study of a microinjection technique for the administration of Infectious Pancreatic Necrosis Virus (IPNV) in fertilized eggs of brook trout Salvelinus fontinalis (Mitchill, 1815) and rainbow brook trout hybrid Salvelinus fontinalis ♂ × Oncorhynchus mykiss ♀. A modular apparatus and technique for the injection of brook trout and rainbow brook trout fish embryos with IPNV are described. The modified microinjection method can be successfully used to administer to the Salmonids eggs infectious substances, simulating the vertical transmission of pathogens.
... Tumors in the swim bladder are reported to be induced in fish via exposure to environmental contaminants and carcinogens 19,20 . In medaka, these tumors are experimentally induced by exposure to N-methyl-N′-nitro-Nnitrosoguanidine (incidence, 0.47%) 21 , 4-chloroaniline (incidence, 17.1%) 22 , and aniline (incidence, 36.7%) ...
... Further, no spontaneous case reports of swim bladder tumors have been published in them. However, in the rainbow trout, swim bladder tumors have been induced by exposure to methylazoxymethanol 19 and dimethylnitrosomorpholine 32 . Additionally, it is possible that the gas glandular epitheliumlike cells are scattered throughout the swim bladder wall in zebrafish 33 . ...
In order to elucidate the effects of swim bladder inflation failure on swim bladder carcinogenesis, we investigated the sequential histopathological changes of swim bladders at 13, 24, 35, and 53 days post-hatch (dph) in medakas with an uninflated swim bladder, which was experimentally induced by denying access to the air-water interface between 0 and 6 dph. The reactive oxygen species (ROS) levels were measured at 24 dph. An uninflated swim bladder was induced in 47.3% of the fish denied access to the air-water interface (the denied group). The total incidence of swim bladder adenoma was 54.1% in the denied group; however, these tumors were observed in all fish with an uninflated swim bladder. In fact, these tumors were observed from 13 dph and onwards. The TBARS levels of the juveniles showed a 2.6-fold increase in fish with an uninflated swim bladder in the denied group compared to that in the control group. It is speculated that swim bladder inflation failure has some effects on the gas gland to produce ROS, leading to DNA damage in the gas glandular epithelium, which develops into swim bladder adenomas. Consequently, it is concluded that denying access to the air-water interface between 0 and 6 dph in medaka is an easy method of inducing swim bladder tumors in a short-term period, and is a useful method for producing tumor-bearing fish.
... Later the model was employed in chemoprevention studies in efforts led by Dr. George S. Bailey (70). There are numerous advantages with this animal model including a low spontaneous background incidence in liver (historically 0.1%), and a high sensitivity to known human carcinogens including aflatoxins, particularly aflatoxin B 1 (AFB 1 , a known human liver carcinogen), polycyclic aromatic hydrocarbons (PAHs), nitrosamines and direct acting carcinogens such as N-methyl-N'nitro-nitrosoguanidine (MNNG) (71,72). Additionally, the low per diem costs for raising trout to 10-12 months of age compared to mice allowed for design of studies employing numbers of animals not achievable in rodent models. ...
... Again, the properties of the model could be exploited to address questions that would be difficult in rodent models. When fed prior to and during carcinogen exposure, I3C was an effective chemopreventive agent against a number of carcinogens (70)(71)(72). Surprisingly, long-term post-initiation feeding of I3C promoted hepatocarcinogenesis in this model (75)(76)(77). Chemoprevention was via the classic "blocking" mechanism resulting from I3C modulation of metabolism. ...
Hydrolysis of glucobrassicin by plant or bacterial myrosinase produces multiple indoles predominantly indole-3-carbinol (I3C). I3C and its major in vivo product, 3,3'-diindolylmethane (DIM), are effective cancer chemopreventive agents in pre-clinical models and show promise in clinical trials. The pharmacokinetics/pharmacodynamics of DIM have been studied in both rodents and humans and urinary DIM is a proposed biomarker of dietary intake of cruciferous vegetables. Recent clinical studies at Oregon State University show surprisingly robust metabolism of DIM in vivo with mono- and di-hydroxylation followed by conjugation with sulfate or glucuronic acid. DIM has multiple mechanisms of action, the most well-characterized is modulation of aryl hydrocarbon receptor (AHR) signaling. In rainbow trout dose-dependent cancer chemoprevention by dietary I3C is achieved when given prior to or concurrent with aflatoxin B1, polycyclic aromatic hydrocarbons, nitrosamines or direct acting carcinogens such as N-methyl-N'-nitro-nitrosoguanidine. Feeding pregnant mice I3C inhibits transplacental carcinogenesis. In humans much of the focus has been on chemoprevention of breast and prostate cancer. Alteration of cytochrome P450-dependent estrogen metabolism is hypothesized to be an important driver of DIM-dependent breast cancer prevention. The few studies done to date comparing glucobrassicin-rich crucifers such as Brussels sprouts with I3C/DIM supplements have shown the greater impact of the latter is due to dose. Daily ingestion of kg quantities of Brussels sprouts is required to produce in vivo levels of DIM achievable by supplementation. In clinical trials these supplement doses have elicited few if any adverse effects. Sulforaphane from glucoraphanin can act synergistically with glucobrassicin-derived DIM and this may lead to opportunities for combinatorial approaches (supplement and food-based) in the clinic.
... lymph nodes and lungs), differences in the lymphatic system, lower body temperatures, the high occur-rence of well-differentiated neoplasms in fishes (Vergneau-Grosset et al. 2017) and the age of animals where neoplasms are usually recognised (Ott Knüsel et al. 2015). Most carcinogen studies were terminated when fish were relatively young and did not reach the age to develop metastases (Bailey et al. 1996). An exception is wild brown bullhead Ameiurus nebulosus, which showed metastases of several histologic types of neoplasia (Spitsbergen & Wolfe 1995). ...
... The prevalence in our study was far higher compared to experimentally induced intestinal adenocarcinoma in rainbow trout and Atlantic salmon, which was 10.62% (Dale et al. 2009), and in zebrafish, where the prevalence ranged from less than 2% (Spitsbergen et al. 2000b, Paquette et al. 2013) to less than 4% (Spitsbergen et al. 2000a). Moreover, rainbow trout seem to be very resistant to intestinal neoplasia (Bailey et al. 1996). ...
Intestinal adenocarcinomas are uncommon in fishes. To date, they have been reported in zebrafish Danio rerio , blue gularis Fundulopanchax sjostedti , koi carp Cyprinus carpio koi , Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss . Metastases are even rarer and have been observed so far at very low prevalence, only in feed-induced adenocarcinoma in Atlantic salmon and rainbow trout. Intestinal adenocarcinoma with liver and heart metastases and mesenteric invasion was found in approximately 33% of 4 yr old rainbow trout from a Slovene hatchery with 2000 breeding trout. During stripping, lumps in the abdominal cavity were palpated in one-third of the breeding fish; some of the fish were anorectic and lethargic, and mortality was slightly increased. Affected trout were euthanized and 4 were submitted for necropsy and histopathology. Necropsy revealed firm, whitish, irregularly lobular masses originating from the intestine. Histologically, the intestinal masses showed a prominent proliferation of tall columnar neoplastic epithelial cells arranged in dense irregular islands or solid areas and papillotubular protuberances. Solid areas of neoplastic cells were also observed in the mesentery of all trout and in the liver of one trout, whereas minute groups of neoplastic cells were seen in the vessels of the intestinal mucosa in all trout and in the myocardium and the liver of one trout. Epithelial origin of neoplastic cells was confirmed by expression of the cytokeratin marker AE1/AE3. The intestinal masses were diagnosed as intestinal adenocarcinoma with mesenteric invasion and metastases to the liver and heart. The cause of intestinal adenocarcinoma was not determined.
... In rainbow trout (Oncorhynchus mykiss) the activation of AFB 1 to AFB 1 -8,9-epoxide is mainly catalysed by CYP 2K1 (Williams and Buhler 1983) and, as in mammals, AFB 1 -N7-Gua is formed and spontaneously converted to AFB 1 -FAPY Croy et al. 1980). Furthermore, AFB 1 is able to induce time-and dose-dependently liver tumours in rainbow trout (Bailey et al. 1996b). This has been documented in at least seven studies (Ayres et al. 1971;Bailey et al. 1987Bailey et al. , 1994Bailey et al. , 1998Dashwood et al. 1989;Lee et al. 1968Lee et al. , 1971 and Table 5 lists the data of five of them. ...
... The rate of repair of AFB 1 -DNA adducts in trout is much slower than in mammals: the "half-life" of the AFB 1 -N7-Gua adduct is 7.5 h in rats, while it is about 21 days in trout ). As noted by Bailey et al. (1996b) the loss of AFB 1 -N7-Gua adducts can be due to chemical conversion, depurination or enzymatic removal and is not a first-order process, so that the above-mentioned values do not reflect true half-lives. However, the long "pseudo half-life" of the AFB 1 -N7-Gua adduct (21 days) in rainbow trout could largely be due to the decreased ability of this animal species to repair bulky DNA adducts. ...
The risk assessment of chemical carcinogens is one major task in toxicology. Even though exposure has been mitigated effectively during the last decades, low levels of carcinogenic substances in food and at the workplace are still present and often not completely avoidable. The distinction between genotoxic and non-genotoxic carcinogens has traditionally been regarded as particularly relevant for risk assessment, with the assumption of the existence of no-effect concentrations (threshold levels) in case of the latter group. In contrast, genotoxic carcinogens, their metabolic precursors and DNA reactive metabolites are considered to represent risk factors at all concentrations since even one or a few DNA lesions may in principle result in mutations and, thus, increase tumour risk. Within the current document, an updated risk evaluation for genotoxic carcinogens is proposed, based on mechanistic knowledge regarding the substance (group) under investigation, and taking into account recent improvements in analytical techniques used to quantify DNA lesions and mutations as well as “omics” approaches. Furthermore, wherever possible and appropriate, special attention is given to the integration of background levels of the same or comparable DNA lesions. Within part A, fundamental considerations highlight the terms hazard and risk with respect to DNA reactivity of genotoxic agents, as compared to non-genotoxic agents. Also, current methodologies used in genetic toxicology as well as in dosimetry of exposure are described. Special focus is given on the elucidation of modes of action (MOA) and on the relation between DNA damage and cancer risk. Part B addresses specific examples of genotoxic carcinogens, including those humans are exposed to exogenously and endogenously, such as formaldehyde, acetaldehyde and the corresponding alcohols as well as some alkylating agents, ethylene oxide, and acrylamide, but also examples resulting from exogenous sources like aflatoxin B1, allylalkoxybenzenes, 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx), benzo[a]pyrene and pyrrolizidine alkaloids. Additionally, special attention is given to some carcinogenic metal compounds, which are considered indirect genotoxins, by accelerating mutagenicity via interactions with the cellular response to DNA damage even at low exposure conditions. Part C finally encompasses conclusions and perspectives, suggesting a refined strategy for the assessment of the carcinogenic risk associated with an exposure to genotoxic compounds and addressing research needs.
... However, a static water bath exposure of rainbow trout (O. mykiss) fry to a solution of 50 ppm MNNG for 30 min produced a high incidence of nephroblastomas (up to 50%) 6 to 9 months later (Bailey, Williams, & Hendricks, 1996). Water bath exposure of approximately 6-10 days old medaka (Oryzias latipes Temminck & Schlegel) and guppy (Poecilia reticulata Peters) fry to methylazoxymethanol acetate (MAM-Ac; Aldrich) for 2 h induced renal nephroblastomas (Hawkins, Overstreet, Fournie, & Walker, 1985). ...
... Birefringent fan-shaped crystals were also observed in the present study. Bailey et al. (1996) mentioned that renal nephroblastomas grow rapidly, become very large and kill the fish through destruction of normal kidney tissue and obstruction of urine flow. This finding suggests that rapid RAHMATI-HOLASOO ET AL. ...
Abdominal distention occurred at an incidence of 1% (15 from 1500 fish) in the population of 1-year-old bester (Huso huso 9 Acipenser ruthenus). Computed tomography (CT) images and radiographs showed a soft tissue mass compressed the posterior part of the swim bladder. Ultrasonography showed that the masses had different patterns. Internal examination revealed the abdominal cavities to be filled with large masses which appeared to encompass most of the visceral organs, including the swim bladder. The masses originated from the posterior kidney. Histologically, the masses were composed of mixtures of embryonal epithelial (tubules and glomeruli), blastema and mesenchymal tissues. The tubules showed cystic, papillary and tubular patterns. Tubules and glomeruloid structures were surrounded by proliferating blastema cells. The primitive mesenchyme was composed of loose streams and whorls of spindle to stellate cells with elongate nuclei. Histological findings in the skeletal muscles, hypoderm and spleen confirmed the metastatic tumour from the kidney in two cases. Immunohistochemically, neoplastic cells of the tubules and glomeruloid structures were positive for cytokeratin AE1/AE3. Sections stained with Masson's trichrome showed blue staining of the stroma. The histopathologic findings were consistent with nephroblastoma.
... Neoplasms of swim bladder origin are rare in fish, with only a few described in a handful of species. Tumours originate from either the epithelial lining of the bladder lumen, epithelial cells of the gas gland, or fibroblastic and smooth muscle tissues of the bladder wall (Bailey, Williams, & Hendricks, 1996;Fournie, Hawkins, & Walker, 1999;Paul et al., 2006). ...
... Proliferative swim bladder lesions, particularly those involving the lining epithelium and gas gland, occur more frequently in fish experimentally exposed to environmental contaminants and carcinogens (Bailey et al., 1996;Brown-Peterson, Krol, Zhu, & Hawkins, 1999). However, spontaneous gas gland hyperplasia, adenomas and adenocarcinomas have been described in control guppies and medaka used in similar experimental studies (Fournie et al., 1999). ...
Neoplasia is rarely reported in syngnathids. This report details an unusual gas gland adenoma in the swim bladder of Hippocampus erectus.
... Rainbow trout is a significant type of salmonid fish from an economic standpoint, and it is used as a model in biological studies. Researchers have dedicated numerous insights to study the morphology, ontogenesis, and function of its immune system (Bailey et al., 1996;Berthelot et al., 2014). Thorgaard et al. (2002) published a review paper specifically dedicated to genomic research on rainbow trout. ...
Lactococcosis caused by Lactococcus garvieae has emerged as one of the most devastating bacterial disease affecting rainbow trout (Oncorhynchus mykiss) worldwide. Understanding the immune response to this streptococcal pathogen is crucial for diagnosing and managing the associated disease and for developing vaccines and preventive measures, such as the selection of naturally resistant trout stocks. Over the past few decades, a significant number of studies have focused on several aspects of lactococcosis, including pathological features, immune responses and vaccination protocols. However, the disease's geographical distribution and its spread over time, the routes of entry and dissemination of the pathogen within the host's body, the progression of infection, and the subsequent inflammatory response of the host still need to be fully explored. This review describes the extant knowledge on pathogenesis, immune response and prevention tools for lactococcosis and identify areas that require further investigations and dedicated studies. Particular emphasis has been placed to describe the current understanding of how immune parameters and inflammatory mechanisms are affected in rainbow trout when they are exposed to natural or experimental infections or undergo vaccination treatments. Ultimately, a more comprehensive understanding of the immune interaction between L. garvieae and rainbow trout will contribute to developing effective disease prevention strategies in aquaculture settings.
... Rainbow trout (Oncorhynchus mykiss) is considered one of the most economically important and promising aquaculture species around the world [37]. In addition, rainbow trout also acts as an excellent model in many research fields, such as physiology, immunology, ecology, and toxicology [38][39][40][41][42]. However, YTH domain-containing genes have yet to be systematically studied up to this point. ...
YTH domain-containing genes are important readers of N6-methyladenosine (m6A) modifications with ability to directly affect the fates of distinct RNAs in organisms. Despite their importance, little is known about YTH domain-containing genes in teleosts until now. In the present study, a total of 10 YTH domain-containing genes have been systematically identified and functionally characterized in rainbow trout (Oncorhynchus mykiss). According to the phylogenetic tree, gene structure and syntenic analysis, these YTH domain-containing genes could be classified into three evolutionary subclades, including YTHDF, YTHDC1 and YTHDC2. Of them, the copy number of OmDF1, OmDF2, OmDF3, and OmDC1 were duplicated or even triplicated in rainbow trout due to the salmonid-specific whole-genome duplication event. The three-dimensional protein structure analysis revealed that there were similar structures and the same amino acid residues that were associated with cage formation between humans and rainbow trout, implying their similar manners in binding to m6A modification. Additionally, the results of qPCR experiment indicated that the expression patterns of a few YTH domain-containing genes, especially OmDF1b, OmDF3a and OmDF3b, were significantly different in liver tissue of rainbow trout under four different temperatures (7 °C, 11 °C, 15 °C, and 19 °C). The expression levels of OmDF1a, OmDF1b and OmDC1a were obviously repressed in spleen tissue of rainbow trout at 24 h after Yersinia ruckeri infection, while increased expression was detected in OmDF3b. This study provides a systemic overview of YTH domain-containing genes in rainbow trout and reveals their biological roles in responses to temperature stress and bacterial infection.
... The acute and subchronic effects in fish include hemorrhage and hepatocyte necrosis. Biliary proliferation, regenerative nodules, and hepatocellular carcinoma are common findings after prolonged exposure (Bailey et al., 1996). ...
... Karaciğere ulaşanlar yağ dokularında depolanabilirler, ya da karaciğer tarafından safra yoluna, ya da böbrekler ve solungaçlar tarafından atılmak üzere kan dolaşımına geri verilebilirler (Ayandiran ve ark., 2009). Atılamayan ağır metallerin ise direkt veya dolaylı yollardan hücresel toksisite, nekroz ve daha sonra doku ölümü olarak ortaya çıkan olaylara sebep olduğu görülmektedir (Bailey, 1996;Jaiswal ve ark., 2018). Ağır metallerce kirletilmiş sularda yaşayan canlılar besin zinciri yolu ile tüketildiklerinde, dolaylı yoldan insan ve hayvan vücuduna girip toksik etkiye neden olabilirler. ...
Ağır metaller yer kabuğunun doğal bileşenlerinden olup, miktar ve dağılımları bölgeden bölgeye farklılık gösterebilmektedir. Doğal yollardan ya da insan kökenli faaliyetlerle deşarj edildiklerinde, yüksek çözünürlüklerinden dolayı son derece toksik kirleticilere dönüşebilmekte ve hava, toprak ve sularda yaşayan canlıları olumsuz etkileyebilmektedirler. Bazı ağır metaller, metabolik aktiviteleri ile ilgili olarak canlı organizmalar açısından eser miktarlarda çok önemliyken bazılarının düşük konsantrasyonları bile canlılarda toksik etkilere sebep olmaktadır. Her ne kadar ağır metallerin çevre ve canlılar üzerine etkileri ağır metalin türüne ve konsantrasyonuna göre değişiklik gösterse de özellikle tarım toprakları ve bitkiler yolu ile besin zincirine girdiklerinde, insanların ve diğer canlıların sağlığı için büyük bir tehdit oluşturmaktadır. Günümüzde ağır metal kirliliğine maruz kalmış toprakların tespit edilmesi ve uygun iyileştirme önlemlerinin geliştirilmesi ile ilgili bazı çalışmalar vardır. Ancak önemli olan konu kirlenmiş alanların temizlenmesi değil, kirliliğin önlenmesi ya da kirlenme hızının minimum seviyeye düşürülmesidir. Ayrıca ağır metallerin çevre ve canlılar üzerindeki etkilerinin iyi anlaşılması gerekmektedir. Bu nedenlerle ağır metallerin tarım toprakları ve sulama suları ile etkileşimlerine ek olarak bitki ve insan sağlığı üzerindeki etkilerinin doğru bir şekilde anlaşılması gerekmektedir. Bu çalışmada; ağır metallerin kaynakları, tarım toprakları ve sulama sularına bulaşma şekilleri, toprak su ve bitkiler üzerindeki etkileri hakkında en son literatürlerden yararlanılarak bilgiler verilmiştir.
... In Oreochromis niloticus, a reduction in genetic damage was observed through the tail length and MG parameters, as has been reported for other bioassays or comet test parameters. Cases where no reduction is observed could be related to poor DNA repair in fish compared to mammals [45][46][47][48]. In Ambystoma mexicanum erythrocytes, it was not possible to evaluate differences between treatments and MGs also showed the ability to detect UV-C-induced genetic damage and post-treatment using AA in erythrocytes from both organisms. ...
The comet assay system is an efficient method used to assess DNA damage and repair; however, it currently provides the average result and, unfortunately, the heterogeneity of DNA damage loses relevance. To take advantage of this heterogeneity, migration groups (MGs) of cell comets can be formed. In this study, genetic damage was quantified in erythrocytes of Oreochromis niloticus and Ambystoma mexicanum exposed to ethyl methanesulfonate (ethyl methanesulfonate (EMS) 2.5, 5, and 10 mM over two hours) and ultraviolet C radiation (UV-C) for 5, 10, and 15 min using the tail length, tail moment, and migration group parameters. Additionally, blood cells were exposed to UV-C radiation for 5 min and treated post-treatment at 5, 10, and 15 mM ascorbic acid (AA) for two hours. With the MG parameter, it was possible to observe variations in the magnitude of genetic damage. Our data indicate that MGs help to detect basal and induced genetic damage or damage reduction with approximately the same efficiency of the tail length and tail moment parameters. MGs can be a complementary parameter used to assess DNA integrity in species exposed to mutagens.
... Among all those varieties Zebrafish was found to be more suitable for investigating the process of embryogenesis, organogenesis, and tumorigenesis. 30 The chemically induced tumors in zebrafish and humans are found to be more similar histopathologically 31 and tumor suppressor genes (TSGs) and orthologous oncogenes have been identified to be similar in Zebrafish and humans. 31 Hepatic gene expression of Zebrafish and humans had shown the conservation of various gene expression profiles in various stages of tumor aggressiveness between these two phylogenetically distinct species [32,33]. ...
Zebrafish is a tropical fresh water fish native of southeast Asia and it measures about 2.5 cm to 4 cm in length. Zebrafish has similar genetic makeup as that of humans. They share 70% of genes with humans. 84% of genes are known to be associated with human disease have a zebrafish counterpart. As a vertebrate, the zebrafish has the same major organs as humans like blood, muscles, kidneys and eyes. The zebrafish genome has been sequenced which enabled researchers to create mutations in more than 14,000 genes to study their functions. Zebrafish has an unique ability to repair their heart muscle researchers working out to find out the specific factors involved in this process so that it can help humans with heart failures. The review throws light on the use of Zebrafish for various biomedical researches.
... Several factors can lead to tumour development in fish. Contaminants and toxins due to human activities, mycotoxins in the environment or in the feed, high amounts of antinutrients in phytogenic feed additives and oncogenic viruses are all capable of inducing the formation of neoplastic masses (Baeverfjord & Krogdahl, 1996;Bailey et al., 1996;Black & Baumann, 1991;Dale et al., 2009). ...
The aquaculture industry is growing and includes the farming and breeding of more than 580 aquatic species worldwide. The rainbow trout (Oncorhynchus mykiss, Walbaum 1792) is the most commonly bred trout species in Hungary. As broodfish form the basis of most fish farms, investigation into tumours occurring in trout, although under‐researched, has proven to be a valuable and necessary field of study. During our investigation, we examined a broodstock of 3‐ to 6‐year‐old rainbow trouts (800) affected with idiopathic intestinal tumours (3%) which had consequentially led to ileus (40%). While performing necropsy, initial pathological observations showed intussusceptions. Tumours were discovered upon opening the body cavity, as well as metastasis forming in the livers and in the vessels of the gills. Histopathological and immunohistochemical tests allowed us to identify the neoplasms. The primary adenocarcinoma was found to have been developed within the intestines of the fish. The tumour tissue broke through the basal membrane and infiltrated the propria, protruding asymmetrically into the lumen of the mid‐intestines, causing it to narrow significantly. This subsequently led to passage disorders, invagination of the intestinal segment and finally the emaciation of the fish. Histopathological and immunohistochemical inspection of the tumour cells displayed a high mitotic index, confirming malignancy.
... Swim bladder tumors can be induced in teleosts via exposure to environmental contaminants and carcinogens 14,15 . Chemically-induced swim bladder tumors originating from gas gland epithelium have been reported in the medaka exposed to N-methyl-N'-nitro-N-nitrosoguanidine 16 , 4-chloroaniline 17 , aniline 17 , or bis(tri-n-butyltin)oxide 18 and in the guppy exposed to methyl mercury chloride 19 . ...
A swim bladder tumor was detected in one scoliotic medaka aged 22 weeks. The tumor was located in the dorsal abdominal cavity, with maximum dimension of 1,850 × 1,500 µm. No swim bladder lumen was identified, and the region where the swim bladder lumen would have been located, was replaced with adipose tissues. The tumor was a non-invasive, expansile, and encapsulated solid mass with a few cysts, and comprised a homogenous population of well-differentiated, densely packed, gas glandular epithelium-like cells. The tumor mass was connected to a rete mirabile that showed a hyperplastic capillary plexus; however, the tumor cells did not invade the rete mirabile, thereby revealing that the tumor was an adenoma originating from the gas glandular epithelium of the swim bladder. Since proliferative lesions have been reported in some teleosts with skeletal deformations, including medaka, the occurrence of a spontaneous swim bladder tumor in teleosts is considered to be closely associated with various types of skeletal deformation, and spinal curvature in particular.
... The species is able to tolerate extreme chemical (contamination) and physical conditions (temperature, salinity, oxygen, etc.) (Hardy Jr, 1978;Bulger, 1984) and is easy to reproduce in captivity. For this reason, mummichog is commonly used in scientific research of stress biology, thermal physiology, toxicology, developmental biology, endocrinology, cancer biology genetics or chronobiology and is considered a model species; it is supposed to be the only freshwater fish species used in a space experiment (Bailey et al., 1996;Hawkins et al., 2003;Law, 2001;Walter & Kazianis, 2001;Winn, 2001;Kent et al., 2009). Gutiérrez-Estrada et al. (1998) state that "F. ...
The mummichog is a cyprinodontiform fish native to eastern coast of North America, where it is very abundant. It is used in the aquarium hobby and for research and could entry through these and other pathways. It is a very hardy species that tolerates a range of temperatures and salinities, has established in two separate areas of the Iberian Peninsula and it is very likely to establish in most coastal areas of the European Union, if introduced. It is rather a sedentary species that has been shown to spread in the Iberian Peninsula although infrequently and slowly. It seems to already impact endemic, endangered Iberian cyprinodontiforms, with less impacts in ecosystem services and reduced economic costs. If introduced to other Mediterranean areas, it is likely to impact other endemic fauna.
... Tumours in fish are nowadays ever more studied to understand aetiology and pathogenesis Marino, Germanà, & Panebianco, 2008;Quaglio et al., 2016). Fish from aquaculture production have a short life span and are unlikely affected by diseases related to senescence, differently from broodstock specimens that have a long life span and are frequently subjected to hormonal stimulation (Bailey, Williams, & Hendricks, 1996). Several fish species are often used as animal models for research Iaria, Migliore, et al., 2019;Iaria, Saoca, et al., 2019;Lo Cascio et al., 2018;Pirrera et al., 2009). ...
Testicular leiomyomas and gonadal dysfunctions are not commonly reported in tel- eost and particularly in broodstock fish. In the present work, a testicular leiomyoma related to an unusual case of spermatogenic failure syndrome, in a broodstock sea- bass coming from an Italian aquaculture farm, is reported. At gross pathology, the circumscribed neoplasia showed several white nodular masses, originating from the gonad, deforming the anatomo-physiological shape. From light microscopy, the pres- ence of neoplastic tissue islands with spindle cells with a whirling pattern and the low presence of connective tissue were evident and originated from smooth muscle cells. Seminiferous tubules showed severe azoospermia with the absence of sperm cells even closer to the neoplastic area. Immunohistochemical analysis was positive for alpha-SMA, desmin and vimentin but negative for S-100, confirming a diagnosis of leiomyoma. This is the first report of spermatogenic failure syndrome associated with a testicular leiomyoma in fish from broodstock. More efforts should be made in studying broodstock fish pathologies related to fish maintenance and hormonal treatments that could economically affect aquaculture production.
... MAM-Ac, a neurotoxin and carcinogen, is a synthetic derivative of cycasin known to induce tumors in the liver, kidney, and small intestine of rats 5,6 and mice 7 . Among teleost fish, MAM-Ac has been known to induce medulloepithelioma in eyes of medaka 8 , pancreatic tumors in guppies 9 , nephroblastomas in guppies and medaka 10 , rhabdomyosarcomas, leiomyosarcomas, fibromas, and neuroblastomas in medaka 10 , and swimbladder tumors, kidney tumors, and glandular stomach tumors in rainbow trouts 11 . The liver is the most common target organ affected by MAM-Ac, and liver tumors have been induced in zebrafish, guppies, mosquitofish 12 , sheepshead minnow, fathead minnow 10 , and medaka 13,14 . ...
We performed a medaka bioassay for the carcinogenicity of methylazoxymethaol acetate (MAM-Ac) to examine the sequential histological changes in the liver from 3 days after exposure until tumor development. The medaka were exposed to MAM-Ac at a concentration of 2 ppm for 24 hours, and were necropsied at 3, 7, 10, 14, 21, 28, 35, 42, 49, 60, and 91 days after exposure. MAM-Ac induced four cases of hepatocellular adenoma and one case of hepatocellular carcinoma in 8 fish after 60 or 91 days of exposure. Histological changes in the liver until tumor development were divided into three phases. In the cytotoxic phase (1–10 days), MAM-Ac-exposed hepatocytes showed vacuolar degeneration and underwent necrosis and apoptosis, resulting in multiple foci of hepatocyte loss. In the repopulation phase (14–35 days), the areas of hepatocyte loss were filled with hepatic cysts and the remaining hepatocytes were surrounded by hepatic stellate-like cells (or spindle cells) and gradually disappeared. In the proliferation phase (42–91 days), the original hepatic parenchyma was regenerated and progressively replaced by regenerative hyperplastic nodules and/or liver neoplasms. The medaka retained a strong hepatocyte regenerative ability in response to liver injury. It is considered that this ability promotes the proliferation of initiated hepatocytes in multistep carcinogenesis and influences the development of liver tumor over a short period in medaka.
... In this frame, we hypothesized that there may be a crosstalk between 17β-estradiol and leptin in the modulation of the 17β-estradiol-induced vtg and consequently of the reproductive responses in relation to the available energy reserves. In order to test this hypothesis, we analyzed the effect of recombinant trout leptin (rt-lep) on 17β-estradiol-induced vtg on liver slices of rainbow trout Oncorhynchus mykiss, one of the most economically relevant species in aquaculture (FAO report 2018) and a widely used laboratory fish model (Bailey et al. 1996;Grunow et al. 2011;Williams 2012). We measured vtg release in the incubation medium, the liver expression of vtg mRNA transcript and lipase activity, and the signal transduction pathways involved in 17β-estradiol-induced vtg synthesis. ...
The existence of nutritional and energy reserves is fundamental for fish female fertility, so that the existence of a correlation between metabolic reserves and reproductive capacity is suggested. Leptin regulates body weight and energy homeostasis. Estradiol induces the synthesis of vitellogenin, a phospholipoglycoprotein produced by the liver and taken up by the growing oocytes. The objective of this study was to investigate the possible existence of a crosstalk between 17β-estradiol (E2) and leptin in the modulation of E2-induced vtg in the rainbow trout Oncorhynchus mykiss. Liver slices were incubated with recombinant trout leptin (rt-lep) at three different concentrations (1–10–100 ng/ml). rt-lep brought about the decrease of E2-induced vtg secretion in the medium and the down-regulation of vtg mRNA expression. Moreover, rt-lep stimulated the lipase activity and diminished the liver fatty acid content. The combined employment of signal transduction inhibitors and the analysis of signal transduction phosphorylated factors revealed that rt-lep effect on E2-induced vtg occurred through the activation of phosphodiesterase, protein kinase C, MAP kinases, and protein kinase A. In conclusion, our study suggests that leptin influences E2-induced vtg synthesis in the rainbow trout Oncorhynchus mykiss by modifying both the protein and the lipid components.
... Fish are frequently used model animals in many fields of research including humane disease modelling (Schartl 2014) and toxicology (Law 2003) with particular focus on small species with rapid maturation and simple maintenance such as zebrafish Danio rerio (Bradford et al. 2017) and medaka Oryzias latipes (Braasch et al. 2015), with hundreds of transgenic and mutant lines. Powerful and versatile fish models provided insights into human disease modelling (Dooley, 2000;Amatruda et al., 2002;Mizgireuv & Revskoy 2006;Beckman, 2007), drug discovery and development (Chakraborty et al. 2009;MacRae & Peterson 2015;Zhu et al. 2016) and toxicology (Bailey et al., 1996;Winn, 2001;Law, 2003;Carvan, 2007). Power of fish models for bioresearch is underlined with their genetic similarity allowing to extrapolate results to humans as well to particularly replace rodent models (Howe et al. 2013). ...
An isogenic line is a group of animals with an identical genotype, and its use is widely accepted for bioresearch standardization. Isogenic lines in fish can be generated within two generations via uniparental inheritance and can facilitate studies for which standardization and consistency are required. The availability and use of isogenic lines is limited, but isogenic lines in some fish species have been generated. Their power has been demonstrated in fields including human disease modelling, drug development, toxicology and also sequencing projects. The genetic basis of desired traits in aquaculture species can be identified using iso-genic lines, which, in turn, will help to improve fish culture. This review summarized knowledge regarding the present status of isogenic lines in fish including approaches for their generation and verification, as well as challenges and potential applications for basic research and aquaculture.
... Heavy metals accumulation, caused the formation of reactive oxygen species that caused changes in metabolism, further leading to cellular intoxication and death at a cellular level. This manifests as necrosis at the tissue level [14]. Mohamed [15], Thophon, Kruatrachue [16], Van Dyk, Pieterse [17] reported that the exposure of fish to pollutants like heavy metals resulted in several pathological alterations in different tissues of fish. ...
Abstract: The present study, pointed to assessment heavy metals accumulation like Aluminum(Al), Cadmium(Cd), Chrome(Cr), Cobalt(Co), Copper(Cu), Iron(Fe), Lead (Pb), Manganese(Mn), Nickel(Ni), Selenium(Se) and Zinc(Zn) in water, sediments and liver of Oreochromis niloticus, which collected from sewage water in El-kharja, New Valley, Egypt using inductively coupled plasma mass spectrometry (ICP-MS). Also, the histopathological changes of liver of Oreochromis niloticus were reported. These histopathological changes were detected using different staining types as a pollution biomarker. Iron showed the highest accumulation level in water (9.06±0.86 ppm), sediments (175.1±20.8 ppm) and liver (158.17±38.59 ppm)., followed by Al> Mn> Ni> Zn> Pb> Cr> Se> Cu> Co> Cd. However, these elements showed Fe> Al> Ni> Mn> Cr> Cu> Zn> Pb> Cd> Se> Co accumulation trend in sediments. In liver these metals showed Fe> Se>Al> Zn>Mn> Cu>Ni> Cr> Pb> Co> Cd trend.
Histopathological examination of fish liver showed signs of progressive alterations such as disorganization of architecture of liver cells, hydropic degeneration and vacuolation of hepatocytes. Also, dilation and congestion in blood sinusoids, hypertrophic and increase in number of küpffer cells were chronicled. Sever deposition of hemosidrin pigments were reported while necrosis with pyknotic nucleus and focal histopathological characters were observed side by side with normal cell.
Our results concluded that water, sediment and liver accumulation toxicity tests and histopathological changes of liver may be associated, and these approaches may be used together to describe the environmental state and water quality assessment.
Keywords: metal accumulation, liver, histopathology, Oreochromis, Sewage water.
... A variety of fishes have been used as model to study tumors induced by environmental carcinogens. Among all the zebrafish proved best for investigating embryogenesis, organogenesis, and impact of environmental carcinogen for the development of cancer [64]. Chemically induced tumors in zebrafish and humans are histopathologically similar [43,65], and orthologous oncogenes and tumor suppressor genes (TSGs) have been identified in fishes and humans [65]. ...
... O. mykiss, in particular, is a useful model for toxicological studies because of its sensitivity to chemicals and because it has been introduced in all kinds of water courses around the world (Bailey et al., 1996;Buhler and Wang-Buhler, 1998). CYP1A catalyzes 7-ethoxyresorufin Odeethylase (EROD), which has become a well-established biomarker of exposure to AhR agonists such as PAH (Clark et al., 2010;Goksøyr and Förlin, 1992;Stegeman and Hahn, 1994). ...
The induction of CYP1A activity (EROD) and protein expression was compared in liver and gills of rainbow trout from a stream polluted with crude oil, and through laboratory exposures to 1% and 5% of water accommodated fraction of the crude oil (WAF) for 1 and 4 days. Gills EROD increased 1.6-2.7-fold in fish from the polluted stream and during experiments, while liver EROD was induced only by 1% WAF at day 1 (1.5-fold). Contrastingly, crude oil pollution strongly induced both liver and gills CYP1A protein expression in the field (14-36-fold) and in experiments (4-25-fold). This highlights that crude oil induced CYP1A activity markedly in gills but only slightly or not at all in the liver, suggesting that differences between organ EROD activities are related to the modulation of CYP1A enzyme activity but not to the regulation at transcriptional or translational levels.
... Rainbow trout sequestrate aflatoxins via a highly efficient microsomal epoxidation, thereby activating the aflatoxins to aflatoxin B 1 exo-8,9-epoxide [37] and expressing little GST activity towards the aflatoxin B 1 exo-8,9-epoxide [38], leading to its high sensitivity to aflatoxins. Additionally, the high responsiveness of rainbow trout to cancer induction might be also related to the poor efficiency of its DNA repair system in removing bulky adducts [39]. Resistant species like Coho salmon, channel catfish and tilapia are less sensitive to aflatoxins because they poorly oxidize aflatoxins and rapidly convert aflatoxins to aflatoxicol that allows for rapid elimination of free aflatoxins [37]. ...
Aflatoxins are fungal metabolites that contaminate foods and feeds, causing adverse health effects in humans and animals. This study determined the occurrence of aflatoxins in fish feeds and their potential effects on fish. Eighty-one fish feeds were sampled from 70 farms and 8 feed manufacturing plants in Nyeri, Kenya for aflatoxin analysis using competitive enzyme-linked immunosorbent assay. Fish were sampled from 12 farms for gross and microscopic pathological examination. Eighty-four percent of feeds sampled tested positive for aflatoxins, ranging from 1.8 to 39.7 µg/kg with a mean of 7.0 ± 8.3 µg/kg and the median of 3.6 µg/kg. Fifteen feeds (18.5%) had aflatoxins above the maximum allowable level in Kenya of 10 µg/kg. Homemade and tilapia feeds had significantly higher aflatoxin levels than commercial and trout feeds. Feeds containing maize bran and fish meal had significantly higher aflatoxin levels than those without these ingredients. Five trout farms (41.7%) had fish with swollen abdomens, and enlarged livers with white or yellow nodules, which microscopically had large dark basophilic hepatic cells with hyperchromatic nuclei in irregular cords. In conclusion, aflatoxin contamination of fish feeds is prevalent in Nyeri, and may be the cause of adverse health effects in fish in this region. Key Contribution: This article is aimed at bridging the gap in knowledge on occurrence, levels of aflatoxins in fish feeds and their potential effects on fish in Kenya. The findings recorded may be used to quantify the aflatoxin burden and justify designing and implementation of control strategies for aflatoxin exposure to fish and contamination of fish feeds.
... Heavy metals are toxic and tend to accumulate in the body organs (El-Ezaby, 1994). It is estimated that fish can act as front-line indicators of suspected aquatic pollutants such as metals (Bailey et aL+ 1996). Fish may absorb dissolved elements and heavy metals from surrounding water and food, which may accumulate in various tissues in significant amounts (Eiman and Zamzam, 1996) and are eliciting toxicological effects at critical targets. ...
... Bainy et al. (1996) noticed oxidative stress in Nile tilapia (Oreochromis niloticus) from a polluted site. Besides acting as a mediator in oxidative stress, higher levels of lipid peroxidation products can adversely affect the cellular functions (Mates, 2000) and adduct with proteins and DNA which may predispose the cell to mutagenesis and carcinogenesis (Bailey et al. 1996). The metabolism of ROS due to their high damaging capacity and biological activity is under fine cellular control and their concentration usually do not exceed 10 -8 m (Halliwell and Gutteridge, 1989). ...
Lipid peroxidation is one of the several physiological mechanisms of the organism by which reactive oxygen species (ROS) may be released which is toxic to cells and tissues. In general, the formation of lipid peroxidation products in tissues may infer the extent of oxidative damage and decline in anti-oxidant defense mechanism. In the present investigation, lipid peroxidation products were measured by determining the malondialdehyde (MDA) concentrations in the tissues of control and exposed fry of Labeo rohita and Cyprinus carpio. The fish fry were exposed to their sub lethal concentration of cadmium (1/5th of 96hrs LC50 i.e., 0.1998ppm for Labeo rohita fry and 4.98ppm for Cyprinus carpio fry respectively) for a period of 20days. Parallel controls were maintained without metal toxicant throughout the experimental period. Samples were collected both from control and exposed fish fry of each species at intervals of 24hrs, 48hrs, 96hrs, 10days and 20days for the estimation of lipid peroxidation products. A maximum amount of lipid peroxidation products was observed at 20 days exposure (3.01n moles of MDA/gm wt of the tissue in Labeo rohita fry and 2.7029 n moles of MDA/gm wt of the tissues in Cyprinus carpio fry). From the data, it was known that more amount of the lipid peroxidation products are found in the fry of Labeo rohita exposed to cadmium than in the fry of Cyprinus carpio indicating the fry of Labeo rohita is more sensitive than Cyprinus carpio.
... Recently, CYP-mediated metabolisms in fish aroused the attention of researchers, and fish were repeatedly evaluated as potential models in drug discovery and toxicology studies (Bailey et al., 1996;Goldstone et al., 2010). Moreover, hepatic CYP in rainbow trout (Oncorhynchus mykiss) was affected by the same flavonoids that affected mammalian CYP, suggesting that rainbow trout can be used for selecting compounds for further research on flavonoiddrug interactions (Burkina et al., 2016b). ...
Phytochemicals are widely present in fruits, vegetables and other plants and have great health benefits owing to their antioxidant properties. They are naturally found in the aquatic environment as well as discharged from sewage treatment plants after their large consumption. Little is known about their impact on fish; particularly in light of their interactions with pharmaceuticals. Therefore, this study was designed to determine the effects of diosmin, naringenin, quercetin and idole-3-carbinol on CYP1A-dependent 7-ethoxyresorufin-O-deethylase (EROD) activity on rainbow trout hepatic microsomes in the presence of two pharmaceuticals: clotrimazole and dexamethasone. The interactions between the phytochemicals and pharmaceuticals used in this study were determined using a combination index. Hepatic microsomes were exposed to two concentrations (1-or 50 μM) of phytochemicals and pharmaceuticals separately and in combinations. Singly, clotrimazole inhibited EROD activity 40% and 90% of control, while dexamethasone did not. Naringenin and diosmin inhibited EROD activity alone up to 90% and 55% respectively, but activities were further inhibited in the presence of either pharmaceutical. The preliminary study of combinations of clotrimazole with phytochemicals primarily showed synergistic effects. While EROD activity was not inhibited in the presence of quercetin or indole-3-carbinol, significant and synergistic inhibition was detected when either of these was combined with clotrimazole or dexamethasone.
... For instance, rainbow trout (Oncorhynchus mykiss) has many advantages as a human carcinogenesis study model. These characteristics include the effects of PAHs (Bailey et al., 1987(Bailey et al., , 1996 and the responses of some genes, such as ras oncogenes (Rotchell et al., 2001). ...
Benzo[a]pyrene (B[a]P) is a petroleum derivative capable of inducing cancer in human and animals. In this work, under laboratory conditions, we analyzed the responses of Colossoma macropomum to B[a]P acute exposure through intraperitoneal injection of four different B[a]P concentrations (4, 8, 16 and 32 μmol/kg) or corn oil (control group). We analyzed expression of the ras oncogene and the Hypoxia-inducible factor-1 alpha (hif-1α) gene using quantitative real-time PCR. Additionally, liver histopathological changes and genotoxic effects were evaluated through the comet assay. Ras oncogene was overexpressed in fish exposed to 4, 8 of 16 μmol/kg B[a]P, showing 4.96, 7.10 and 6.78-fold increases, respectively. Overexpression also occurred in hif-1α in fish injected with 4 and 8 μmol/kg B[a]P, showing 8.82 and 4.64-fold increases, respectively. Histopathological damage in fish liver was classified as irreparable in fish exposed to 8, 16 and 32 μmol/kg μM B[a]P. The genotoxic damage increased in fish injected with 8 and 16 μmol/kg in comparison with the control group. Acute exposure of B[a]P was capable to interrupt the expression of ras oncogene and hif-1α, and increase DNA breaks due to tissue damage.
... The CYP2 family of enzymes is represented by CYP2K1 and CYP2M1 in rainbow trout (Buhler et al. 1994, Yang et al. 1998. CYP2K1 is the most abundant constitutively expressed hepatic cytochrome P450 found in sexually immature rainbow trout and it shows an unusually high sensitivity to carcinogens (Bailey et al. 1996). CYP2K1 has benzphetamine N-demethylase and steroid hydroxylase activities, and both CYP2K1 and CYP2M1 are involved in lauric acid metabolism and some longer fatty acids in rainbow trout (Yang et al. 1998 and both are involved in the metabolism of estrogenic hormones Wang-Buhler 1998, Buhler et al. 2000). ...
Cytochrome P450 (CYP) gene expressions were studied by semi-quantitative polymerase chain reaction (PCR) to assess oil pollution using liver and gills of juvenile rainbow trout Oncorhynchus mykiss sampled from La Mina stream (Bariloche, Argentina), up and downstream of a discharge of petroleum spill from an unproductive wellbore. Data was complemented with laboratory exposure to water accommodated fraction (WAF) from the oil spill. Oil exposure induced gill and liver CYP1A transcription (1.2-1.4-fold), which was used as reference biomarker. In both organs, CYP2K1 transcript was induced after 5 % WAF exposure, 24 h (1.8-2.0-fold), and it was down-regulated in more extended time (1.2-1.5-fold). CYP2M1 transcript levels were down-regulated in liver of impacted wild trout and in trout exposed to 5 % WAF during 96 h (1.5-1.7-fold). CYP2K1 and CYP2M1 are involved in the metabolism of fatty acids and estrogenic hormones, and its down-regulation would indicate metabolic alterations. CYP3A27 levels were only induced in liver of wild trout (1.6-fold), possibly related to crosstalk between aryl hydrocarbon receptor and pregnane X receptor signaling pathway. While CYP1A mRNA expression appears to be a more robust biomarker for detecting the effects of crude oil under different dose and exposure time, the expression of CYP2K1, CYP2M1 and CYP3A27 mRNAs would indicate different time exposure to oil pollution, particularly under high oil concentration in water. Hence, patterns of multiple CYP genes expression in rainbow trout gills and liver could be especially valuable for monitoring programs of crude oil pollution and remediation actions on Patagonian freshwater ecosystems.
... In this study, exposure of H. bidorsalis to Paraquat at the various concentrations induced an increase in MDA levels in the liver, kidney and the gills. These increases show the enhancement of LPO and suggest that ROS may be involved in the metabolism of Paraquat leading to peroxidation membrane lipids of these organs which can lead to loss of cellular function and mutations (Munkittrick et al., 2000; Bailey et al., 1996). Increase lipid peroxidation recorded in this study has also been reported in several fish species exposed to other herbicides by Oropesa, et al. (2009), Elia et al. (2002) and Farombi et al. (2008The changes in enzyme activity during this study were dependent on the herbicide concentration. ...
The study assessed oxidative stress and antioxidant response parameters in the liver, kidney and gills of the Giant
African catfish; Heterobranchus bidorsalis. Fish were exposed to different Paraquat concentrations of 0.0mg L-1,
0.69mg L-1, 1.37mg L-1 and 2.75mg L-1 for 28 days. Oxidative stress (Lipid peroxidation) was measured as
malondialdehyde (MDA). Antioxidant response was measured as superoxide dismutase (SOD), catalase (CAT)
and glutathione peroxidase (GH-Px). The herbicide stress caused significant (P<0.05) increase in the MDA, SOD
and CAT activity in all the organs examined except in the kidney in which the CAT was significantly decreased.
The GH-Px activity, decreased in these organs and the changes in all these parameters were concentration
dependent. These parameters significantly differ from the control (0.0mg L-1) with no paraquat concentration.
The study suggests that Paraquat induces oxidative stress in exposed fish. This antioxidant response has the
potential to serve as a biomarker for the monitoring of bipyridyl herbicides in aquatic environments.
Model organisms are responsible for many important discoveries and advancements in science. This group of species shares a suite of traits making them uniquely suited to be models including being easy to maintain and genetically manipulated within a laboratory setting. Aquatic model organisms are specially positioned for ecotoxicity assessment because of their ability to serve as predictors of toxicity for other organisms, as well as sentinel species for environmental health. The standards of regulatory toxicity assessment and the organisms used in these assays are dictated by environmental agencies. A diversity of freshwater, estuarine, and marine algae, invertebrates, and fish are currently used as model species for ecotoxicity assessment. The popularity of model organism research has created a bias for those studies and limited the diversity of the data produced. However, the expansion of genomic technologies has opened the door to a host of emerging and alternative model organisms. Therefore, though, historically, the number of ecotoxicity model organisms has been limited, the definition is expanding to include a broader range of well-studied organisms.
Neoplasia in elasmobranchs is uncommonly documented. This report describes primary hepatic neoplasia in three adult female bamboo sharks under managed care: biliary adenocarcinoma in a whitespotted bamboo shark (Case 1; Chiloscyllium plagiosum), biliary adenocarcinoma in a brownbanded bamboo shark (Case 2; Chiloscyllium punctatum), and hepatocellular carcinoma in a whitespotted bamboo shark (Case 3). Case 1 presented with extensive cutaneous papillomas and was electively euthanized. At necropsy, a 4-cm-diameter, pale-tan, firm hepatic mass was identified and diagnosed histologically as a biliary adenocarcinoma. Case 2 presented with decreasing body condition despite normal food intake. Coelomic ultrasound and exploratory surgery revealed several large masses in both hepatic lobes, and the patient was euthanized. At necropsy, nine, 1-6-cm-diameter, black to tan, firm hepatic masses were identified and diagnosed histologically as biliary adenocarcinoma with branchial intravascular neoplastic emboli. Case 3 presented for routine health examination and was euthanized for diagnostic purposes after coelomic ultrasound revealed multiple hepatic masses. At necropsy, two 1-3-cm-diameter, brown- and-black mottled, firm hepatic masses were identified and diagnosed histologically as hepatocellular carcinoma. Immunohistochemistry was performed in two of these cases and was noncontributory, likely because of a lack of cross reactivity between antibodies (antipancytokeratin) and elasmobranch tissues.
There have been many reports of cancer or neoplastic diseases in wild fish populations that have a chemical carcinogenic causation. Epidemics of hepatic neoplasia occur in at least 16 species of fish from 25 different polluted fresh and marine water habitats. Fish that live in carcinogen-polluted sediments, that are older and bottom dwellers tend to develop tumors over a period of time because carcinogenesis is a type of chronic toxicity. Field studies comparing tumor rates between contaminated and relatively clean aquatic regions produce the observation that as contamination levels increase or decrease, the tumor rates in fish follow in a direct relationship. This discussion will briefly highlight the range of chemically induced cancers in fish focusing on the pathogenesis and risk factors.
Fish are useful animal models for studying effects of nutrients and environmental factors on gene expression (including epigenetics), toxicology, and carcinogenesis. To optimize the response of the animals to substances of interest (including toxins and carcinogens), water pollution, or climate changes, it is imperative to understand their fundamental biochemical processes. One of these processes concerns energy metabolism for growth, development, and survival. We have recently shown that tissues of hybrid striped bass (HSB), zebrafish, and largemouth bass (LMB) use amino acids (AAs; such as glutamate, glutamine, aspartate, alanine, and leucine) as major energy sources. AAs contribute to about 80% of ATP production in the liver, proximal intestine, kidney, and skeletal muscle tissue of the fish. Thus, as for mammals (including humans), AAs are the primary metabolic fuels in the proximal intestine of fish. In contrast, glucose and fatty acids are only minor metabolic fuels in the fish. Fish tissues have high activities of glutamate dehydrogenase, glutamate–oxaloacetate transaminase, and glutamate-pyruvate transaminase, as well as high rates of glutamate uptake. In contrast, the activities of hexokinase, pyruvate dehydrogenase, and carnitine palmitoyltransferase 1 in all the tissues are relatively low. Furthermore, unlike mammals, the skeletal muscle (the largest tissue) of HSB and LMB has a limited uptake of long-chain fatty acids and barely oxidizes fatty acids. Our findings explain differences in the metabolic patterns of AAs, glucose, and lipids among various tissues in fish. These new findings have important implications for understanding metabolic significance of the tissue-specific oxidation of AAs (particularly glutamate and glutamine) in gene expression (including epigenetics), nutrition, and health, as well as carcinogenesis in fish, mammals (including humans), and other animals.
Non-mammalian vertebrates including birds, fish, and amphibians have a long history of contributing to ground-breaking scientific discoveries. Because these species offer several experimental advantages over higher vertebrates and share extensive anatomic and genetic homology with their mammalian counterparts, they remain popular animal models in a variety of fields such as developmental biology, physiology, toxicology, drug discovery, immunology, toxicology, and infectious disease. As with all animal models, familiarity with the anatomy, physiology, and spontaneous diseases of these species is necessary for ensuring animal welfare, as well as accurate interpretation and reporting of study findings. Working with avian and aquatic species can be especially challenging in this respect due to their rich diversity and array of unique adaptations. Here, we provide an overview of the research-relevant anatomic features, non-infectious conditions, and infectious diseases that impact research colonies of birds and aquatic animals, including fish and Xenopus species.
Organophosphate and organochlorine pesticides are banned in most countries because they cause high toxicity and bioaccumulation in non-target organisms. Pyrethroid pesticides have been applied to agriculture and aquaculture since the 1970s to replace traditional pesticides. However, pyrethroids are approximately 1000 times more toxic to fish than to mammals and birds. Fish-specific organs such as the gills and their late metabolic action against this type of pesticide make fish highly susceptible to the toxicity of pyrethroid pesticides. Oxidative stress plays an important role in the neurological, reproductive, and developmental toxicity caused by pyrethroids. Deltamethrin, cypermethrin, and lambda-cyhalothrin are representative pyrethroid pesticides that induce oxidative stress in tissues such as the gills, liver, and muscles of fish and cause histopathological changes. Although they are observed in low concentrations in aquatic environments such as rivers, lakes, and surface water they induce DNA damage and apoptosis in fish. Pyrethroid pesticides cause ROS-mediated oxidative stress in fish species including carp, tilapia, and trout. They also cause lipid peroxidation and alter the state of DNA, proteins, and lipids in the cells of fish. Moreover, changes in antioxidant enzyme activity following pyrethroid pesticide exposure make fish more susceptible to oxidative stress caused by environmental pollutants. In this review, we examine the occurrence of pyrethroid pesticides in the aquatic environment and oxidative stress-induced toxicity in fish exposed to pyrethroids.
Background: Rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) is one of the most commonly bred fish species in Europe, which plays an important role in the gastronomy. The brood stock has a significant value at the farms, therefore the prevention of diseases is crucial. Objectives: During our research we investigated an idiopathic intestinal tumour in rainbow trout. All the examined individuals were 3-6 years old, as tumours need a longer period to develop and therefore cannot be found in fingerlings, summerlings and consumable fish. The healthcare of the valuable broodfish is of great importance in fish farms. Nowadays, the investigation of tumours growing in trouts is considered a pioneering field of research. Materials and Methods: During the necropsy, tumours found on the gills and in the intestines of broodfish were identified with the help of histological and immunohistochemical tests. Samples from the organs (stomach, intestines, liver, gills) were put into 8% buffered formalin for fixation. Nikon Optiphot-2 microscope was used to examine the sectioned slides. The indirect immunohis-tochemical method was applied with Ventana immunohistochemistry stainer. Results and Discussion: The primary adenocarcinoma developed in the small intestine. The malignant tumour tissue with a high mitotic index infiltrated and broke through the basal membrane and the propria. The adenocarcinoma spreads asymmetrically to the cross-section of the intestine narrowing the lumen of the intestine causing passage disorder of the intestinal contents and emaciation in the fish. The primary tumour formed metastases in the vessels of the liver and gill, which were also found in the middle and peripheral part of the primary lamellae, resulting in respiratory disorder. The intestinal carcinoma of the trout gave positive results to pan-cytokeratin and E-cadherin in immu-nohistochemical tests, the peritumoral endothel cells also showed claudin-5 positivity. The other tests with anti-vimentin, anti-alfa-smooth muscle actin, anti-S-100 protein, anti-NSE antibody led to negative results. Identification of gastro-intestinal tumours in rainbow trout (Oncorhynchus mykiss Walbaum, 1792)
The liver hosts numerous vital functions, such as biotransformation and excretion of xenobiotics. Synthetic oestrogens influence liver structure and function, leading to adaptations or to dysfunctions/injury. They are often stated to induce increases in fish liver weight, but there is controversy regarding how: if by changes in hepatocyte size (hypertrophy) and/or number (hyperplasia). Using platyfish as the experimental model, our primary aim was to assess if/how hepatocytes reacted to a sub-acute oestrogenic exposure. A complementary aim was to generate fundamental structural data for the liver of that model organism. Adult males were injected intramuscularly with 17α-ethinylestradiol (EE2) (25 μg/g), every 72 h for two weeks. Control fish were given solvent only. Body and liver morphometry were registered, and hepatocytes examined through histology and stereology at light microscopy. Immunohistochemistry evaluated hepatocytic vitellogenin (VTG) content. Treated and control fish did not differ as to quantitative parameters. Nevertheless, exposed fish were sensitive to EE2. VTG tagging was positive in their hepatocytes and these tended to be more basophilic, though not fully oestrogenized. We hypothesise that the platyfish liver is not particularly sensitive to the disrupting action of EE2 because of its reproductive mode; with no production peaks of VTG and no huge changes in endogenous sex-steroids. The fish may have had no evolutionary pressure for hepatocytes to be particularly reactive to oestradiol (E2). In the end, this study offers the first unbiased estimation of the liver cellularity in the platyfish, as well of the hepatocytic volume, serving now as a baseline reference.
Background: The authors examined a marbled sailfin pleco (Pterygoplichthys joselimaianus Weber, 1991) brought to the clinic of the Department of Exotic Animal and Wildlife Medicine of the University of Veterinary Medicine in September 2016. The owner purchased the animal in January 2016 in a special aqua-ristic shop. The owner kept the fish in a 350 I tropical aquarium with other small sized fish species (Paracheirodon innesi, Danio margaritatus). Objectives: During the physical examination there was a lentil-sized papillo-ma-like tumour in the right corner of the mouth. Due to the increasing growth rate of the tumour the veterinary team of the clinic decided to perform removal surgery on the fish. The authors used the removed tissue to perform histo-pathological examination to identify the type of the tumour. Materials and Methods: The veterinary team examined the fish and prepared the anaesthesia bath. The bath contained 0,170 mg/ml MS-222 (tricaine meth-anesulfonate), an anaesthetic commonly used in fish veterinary procedures. After removing the tumour, the wound was treated with Betadine ointment. The tissue was fixed for 24 hours in buffered formalin. After the fixation the sample was infiltrated and embedded into paraffin blocks. Leica manual microtome was used for section cutting. The prepared histological slides were stained with haematoxylin and eosin and were examined with Olympus CX21 microscope. The identified tumour was compared to neoplasms found in other ornamental fish species described earlier in literature review. Results and Discussion: After the results of the histopathological examination the tumour was identified as a papilloma. According to the authors' hypothesis the tumour was caused by a mechanical trauma or an injury. Nowadays in aqua-ristic owning high-priced fish species is increasing in popularity. However, these valuable species require adequate veterinary supervision, prevention, diagnostics and treatment as well.
Anxiety is a widespread psychiatric disorder. The search for a cure is still continuing since many of the synthetic drugs were inefficient in completely treating anxiety, yet caused some dangerous side effects until many of the drugs were withdrawn from the market. One promising source of new anxiolytics could be herbal medicines. The challenge is to screen plant extracts. Rodent models can be used for this purpose but are expensive. Moreover, rodent tests are costly and consume relatively large quantities of sample. For this reason, alternative animal models may be useful. Zebrafish larvae have many advantages for screening natural products. The main advantage is that they can be produced cheaply and in large numbers. Several studies have shown that the zebrafish is a good model for studying drugs that affect anxiety. This review focuses on the use of animal models, including zebrafish larvae, for studying anxiety and screening for herbal medicines that modulate anxiety. Finally, future prospects of the zebrafish larva as an alternative model in this field are also discussed.
Indole-3-carbinol (I3C) is a secondary plant metabolite produced in vegetables of the Brassica genus, including cabbage, cauliflower, and brussels sprouts. I3C is both an anti-initiator and a promoter of carcinogenesis. Consumption of I3C by humans and rodents can lead to marked increases in activities of cytochrome P-450-dependent monooxygenases and in a variety of phase II drug-metabolizing enzymes. We have reported previously that the enzyme-inducing activity of I3C is mediated through a mechanism requiring exposure of the compound to the low-pH environment of the stomach. We report here the aromatic hydrocarbon responsiveness-receptor Kd values (22 nM-90 nM), determined with C57BL/6J mouse liver cytosol and the in vitro- and in vivo-molar yields (0.1-6%) of the major acid condensation products of I3C. We also show that indolo[3,2-b]carbazole (ICZ) is produced from I3C in yields on the order of 0.01% in vitro and, after oral intubation, in vivo. ICZ has a Kd of 190 pM for aromatic hydrocarbon responsiveness-receptor binding and an EC50 of 269 nM for induction of cytochrome P4501A1, as measured by ethoxyresorufin O-deethylase activity in murine hepatoma Hepa 1c1c7 cells. The binding affinity of ICZ is only a factor of 3.7 x 10(-2) lower than that of the highly toxic environmental contaminant and cancer promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin. ICZ and related condensation products appear responsible for the enzyme-inducing effects of dietary I3C.
The harmful effects of Aflatoxin B1 (AFB1) are a consequence of it being metabolized to AFB1-8,9-epoxide, a compound that serves as an alkylating agent and mutagen. The toxicity of AFB1 towards different cells varies substantially; sensitivity can change significantly during development, can be modulated by treatment with xenobiotics and is decreased markedly in preneoplastic lesions as well as in tumors. Three types of resistance, namely intrinsic, inducible and acquired, can be identified. The potential resistance mechanisms include low capacity to form AFB1-8,9-epoxide, high detoxification activity, increase in AFB1 efflux from cells and high DNA repair capacity. Circumstantial evidence exists that amongst these mechanisms the glutathione S-transferases, through their ability to detoxify AFB1-8,9-epoxide, play a major role in determining the sensitivity of cells to AFB1.
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.
Butylated hydroxyanisole (BHA) and β-naphthoflavone (BNF), both chemicals with anti-carcinogeneic properties in some experimental
animals, were compared for effects on afiatoxin B1 (AFB1) metabolism, hepatic DNA adduct formation and carcinogenesis in the
rainbow trout. Dietary BHA had no effect on the hepatic tumor incidence when fed at 0.03 or 0.3% 4 weeks prior to and during
a 4 week dietary exposure of 10 p.p.b. AFB1. BNF, when fed at 0.005 or 0.05% under similar conditions, significantly reduced
tumor response, which confirms previous results in trout (Nixon et al.9 Carcinogenesis, 5, 615–619, 1984). BHA fed at either 0.03 or 0.3% for 8 weeks had no post-initiation effect on the 52 week hepatic tumor
incidence of trout exposed to a 0.5 p.p.m. AFB1 solution as embryos. A similar post-initiation exposure to 0.05% BNF significantly
enhanced AFB1 tumor response. The influence of dietary BHA and BNF on AFB1 metabolism and DNA adduct formation and persistence
in trout were examined. A 3 week pre-treatment with 0.3% dietary BHA had no effect on in vivo hepatic nuclear AFB1-DNA adduct formation at 0.5, 1, 2 and 7 days after AFB1 i.p. injection. By contrast 0.05% dietary BNF
reduced hepatic AFB1-DNA adducts to 33–60% of control levels at 0.5, 1, 2 and 4 days after AFB1 exposure. This was accompanied
by significantly lower blood and liver levels of AFB1 during the first 24 h after i.p. injection. Livers of BNF trout also
contained 4-fold more of the less carcinogenic metabolite, aflatoxin M1, and 50% less aflatoxicol (AFL), a metabolite with
similar carcinogenicity as AFB1. Bile AFL-glucuronide levels were significantly decreased in BNF-fed trout, but total bile
glucuronides were significantly increased due to a 15-fold increase in aflatoxicol-M1 glucuronide. Freshly isolated hepatocytes
from BHA-fed fish, when incubated with AFB1 for 1 h, showed no difference in levels of AFB1-DNA adducts or ratios of AFB1
metabolites when compared to hepatocytes isolated from fish fed a control diet only. By contrast, dietary BNF has been previously
shown to greatly enhance AFM1 production, reduce AFL production, and significantly reduce AFB1-DNA adduct formation in isolated
trout hepatocytes (Bailey et al., Natl. Cancer Inst. Monograph, 65, 379–385, 1984). These results indicate that dietary BHA up to 0.3% does not alter AFB1 metabolism or DNA adduction in
trout, nor does it inhibit or promote AFB1 hepatocarcinogenesis in this species. This is in contrast to anti-oxidant enhancement
of AFB1-glutathione conjugation, reduction of AFB1-DNA binding, and consequent reduction of tumor response in rats. The null
results in trout thus support enhanced glutathione conjugation as the major mechanism for BHA inhibition of AFB1 cardnogenesis
in mammalian models. By contrast, BNF dietary pre-treatment appears to inhibit AFB1 carcinogenicity in trout by enchancing
glucuronide formation and elimination of the carcinogen, leading to reduced DNA adduct formation in target tissue.
Indole-3-carbinol (I3C), a secondary metabolite from cruciferous veg etables, inhibits aflatoxin li, (AM!,) hepatocarcinogenesis in trout (Bailey et al., J. Nati. Cancer Inst., 78: 931-934, 1987) and rats (Selivonchick a a/., unpublished results) when given prior to and with carcinogenbut promotes carcinogenesis in both species when given continuously follow ing AFBi initiation. Since human I3C intake may not be continuous, and the promotional stimulation may be reversible, we have assessed I3C promotion using delayed and discontinuous exposure protocols. Following initiation with AFBi, I3C was fed to trout for varying periods of time, with varying lengths of delay after initiation and continuous or intermit tent patterns of I3C treatment. Promotional enhancement of tumor inci dence by I3C was found to be significant when I3C treatment was delayed for several weeks or months after the initial AI H, challenge. Promotion also was found to increase with length of exposure to I3C treatment and to be decreased but still evident when I3C was given in alternating months or weeks, or twice per week only. These results do not support the idea that promotional stimulation in hepatocarcinogenesis is a reversible phenomenon. To quantify I3C promotional potency in terms of its dietary concentra tion, a series of \ 1H, tumor dose-response curves was established, each with a different level of I3C fed continuously following Al H, initiation. The resultant tumor dose-response curves, plotted as logit percentage of incidence versus log \l H, dose, were displaced parallel toward lower AFBi 50% tumor take (II ).-,>) values with increasing I3C concentration. The level of I3C that halves the AFBi dose for 50% tumor incidence was calculated to be approximately 1000 ppm I3C, fed continuously, with no substantial threshold for promotion. By comparison, I3C, when fed before and with AFBi, shows a 50% inhibitory value (I3C concentration that doubles the dose of AFBi for 50% tumor incidence) in trout of 1400 ppm I3C IDashwood el at., Carcinogenesis (Lond.), 10:175-181, 1989|. Thus the potential for I3C as a dietary additive to promote prior hepatic initiation events when fed continuously is approximately as great as its potential to inhibit concurrent AFBi initiation.
Male rainbow trout kidney (Oncorhynchus mykiss) during the late reproductive stage displays considerably higher levels of cytochrome P450 than that of the female. Two P450 forms, with molecular weights of P54 000 and 52 000 (designated P450 KM1 and KM2, respectively), were partially purified from the male kidney. Specific antibodies were raised against the P450 KM2 form and used with a Western blot technique to demonstrate the virtual absence of this P450 form in female and juvenile kidney microsomes. Treatment of juvenile trout with androgens (11-ketotestosterone being the most potent) increased the renal cytochrome P450 content as well as progesterone 16χ-hydroxylase and ethoxycoumarin-O-deethylase activities. Androgen treatment of juvenile trout also induced the P450 KM2 in the kidney, as revealed by Western blotting. Thus, the male rainbow trout kidney during the late reproductive stages expresses at least one sex-specific P450 form which is regulated by circulating androgens.
Incidental observations of Atlantic tomcod during routine laboratory processing revealed that a portion of the adult population collected during the 1977–78 spawning season had enlarged livers containing dark coloured tumours and other abnormalities. Of the total of 264 livers collected between 16 January and 27 February 1978 and grossly examined for prevalence of abnormalities, 25% appeared to contain neoplastic nodules and hepatocellular carcinoma. One liver contained a massive tumour (7 × 12 mm) that involved approximately 60% of the liver. The exact causes of the high prevalence of hepatocellular carcinoma are unknown but poly-chlorinated biphenyls (PCBs) are suspected of having a possible role. The Hudson River is known to contain elevated concentrations of PCBs. Twelve tomcod livers from the 1977–78 spawning population representing both normal and hepatoma conditions contained concentrations of PCBs ranging from 10–9 to 98–2 ppm (mean of 37–5 ppm).
We undertook this study to answer several questions regarding nitrosamine metabolism. Kinetics of nitrosamine metabolism showed the involvement of at least two enzymes in the dealkylation of N-nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA) in mouse liver microsomes. Coumarin inhibited both reactions competitively. On the other hand, microsomal coumarin 7-hydroxylase was inhibited by NDMA (Ki 2.7 mM) and NDEA (Ki 0.013 mM). The big difference in the Ki values suggests a higher affinity of NDEA than NDMA to Cyp2a-5 (mouse cytochrome P450coh). A specific antibody against Cyp2a-5 inhibited more of the microsomal NDEA (up to 90%) than NDMA (up to 40%) dealkylation. The converse was true with anti-Cyp2e-1 antibody. These results suggest that the primary substrate for Cyp2a-5 is NDEA and for Cyp2e-1, NDMA. Western blot analysis of human liver microsomes showed a great interindividual variation in the amounts of CYP2A6 (human cytochrome P450coh) and CYP2E1. Also, courmarin 7-hydroxylation and nitrosamine dealkylation varied greatly among individuals. A high correlation (r = 0.93, P < 0.001) was found between NDEA and coumarin metabolism. Both activities were associated with CYP2A6. On the other hand, little or no correlation was found between microsomal CYP2A6 and CYP2E1 or between CYP2E1 and NDEA dealkylation. Immunoinhibition of human microsomal NDEA metabolism by CYP2a-5 antibody varied greatly among individuals (10–90%), suggesting, as in the case of mice, that NDEA is metabolized primarily by CYP2A6, at least in some individuals. Taken together the data suggest that (1) the metabolic activation of nitrosamines in humans varies greatly among individuals; (2) different nitrosamines may partially be metabolized by different cytochrome P450 isozymes; and (3) because of similarities between nitrosamine metabolism in mice and humans, inbred strains of mice would be relevant experimental models for studying nitrosamine activation.
Neoplastic pigment cells can be induced by N-methyl-N-nitrosourea (MNU) in certain genotypes of the fish Xiphophorus that never develop such cells spontaneously. In some, the neoplastic cells retain their incompletely differentiated stage and proliferate, apparently due to unrestrained division, into malignant pigment cell neoplasms (‘melanomata’). In other fish most of the neoplastic pigment cells become terminally differentiated and form harmless ‘hyperplastic foci’. Terminal differentiation of MNU-induced neoplastic pigment cells appears to be predominantly controlled by a gene complex designated ‘differentiation’ (Diff).
The cytochrome P450 monooxygenase system metabolizes a large number of xenobiotic compounds including many environmental pollutants. This metabolism can lead to detoxification, or in some cases, activation to reactive intermediates with toxic and carcinogenic effects. Among animals living in the aquatic environment the cytochromes P450 have been studied best in fish. The catalytic properties and factors regulating their activity have been an important field for aquatic toxicology investigations. A major concern is the mechanisms associated with the induction of cytochrome P450 by organic hydrocarbons. Several variables such as development stage, sex, and ambient temperature may influence the P450 enzyme system and its inductive response. The objective of this review is to discuss recent progress in studies of regulation of the cytochrome P450 enzyme system in fish.
In fish, as well as in mammals, it is well known that the cytochrome P450-dependent oxidative metabolism of xenobiotics can generate DNA-reactive species. Moreover, this metabolism is known to be inducible by several compounds of environmental significance, such as polychlorobiphenyls, polycyclic aromatic hydrocarbons (PAHs) and dioxins. Consequently, we studied the relationship between the degree of induction of the cytochrome P4501A, expressed as that of 7-ethylresorufin O-deethylase (EROD) activity, and the level of DNA-adducts, using the post-labelling assay, in the liver of rainbow trout exposed to benzo(a)pyrene (a representative PAH). The results showed a significant 2- to 4-fold increase in EROD activity 2, 4 and 8 days after treatment, paralleled by an increase in DNA-adduct levels. This work further emphasizes the involvement of cytochrome P4501A in the metabolism of benzo(a)pyrene into genotoxic metabolites in rainbow trout.
Liver tumors were induced in rainbow trout (Salmo gairdneri) 1 yr after trout were injected with aflatoxin B1 in the sac-fry stage of development. In this modification of the trout embryo microinjection carcinogenesis assay, there are fewer mortalities than in earlier embryo stage injection protocols. Injection efficiency, as measured by retention of [14C]benzo[a]pyrene in the embryo, and sensitivity to the carcinogenic effects of aflatoxin B1 is not reduced by injection at this later stage of development.
Combined field and laboratory studies were carried out to assess the possible role of contaminated bottom sediments to neoplastic disease in fish from eastern Lake Erie and the upper Niagara River. Correlations between sediment polycyclic hydrocarbons, neoplasms in feral fish, and the induction of neoplasms in bullheads (Ictalurus nebulosus) by exposure to extracts of polluted sediment support the hypothesis that some fish neoplasms result from exposure to carcinogenic chemicals present in the fishes environment.
The carcinogenic effects of 7,12-dimethylbenz[a]anthracene (DMBA) on the king cobra strain of guppy (Poecilia reticulata) were examined. Specimens were exposed for 6 h once weekly for 4 wk to DMBA. Guppies were 6–11 days old at the initial exposure. Treatment groups included the following: (1) untreated control; (2) dimethylformamide (DMF) carrier control; (3) low, water-mediated DMBA concentration (about 1–3 μg/l or ppb DMBA); (4) intermediate, DMF-mediated DMBA concentration (about 20 ppb DMBA); and (5) high, DMF-mediated DMBA concentration (about 35 ppb DMBA). Hepatic neoplasms developed in guppies exposed to the intermediate and high concentrations. Both of these exposure media contained the same concentrations of soluble DMBA. The high exposure medium, however, also contained an insoluble, particulate fraction of DMBA. Hepatic neoplasm incidences in fish exposed to the intermediate concentrations were 10% at 24 wk and 19% at 37 weeks after the initial exposure. In samples from the high concentration group, 47% had hepatic neoplasms at 24 weeks and 46% at 37 wk. Histologically, the hepatic lesions were categorized as altered foci, hepatocellular adenomas, and hepatocellular carcinomas. In addition to liver neoplasms, several other types of lesions developed in DMBA-exposed guppies. These included two undifferentiated sarcomas, a rhabdomyosarcoma, a renal adenocarcinoma, and a neurilemmoma. Only one control specimen (representing 0.38% of all controls) had a neoplastic lesion, a small hepatocellular adenoma in a 37-wk specimen. This study demonstrates that the guppy develops hepatic and extrahepatic neoplasms rapidly following brief water-borne exposures to those rather low levels of DMBA. Furthermore, the study showed that the particulate fraction of DMBA contributes to the carcinogenicity of the compound.
The major constitutive cytochrome P-450 isoenzyme in rainbow trout, denoted cytochrome P-450con was studied. Western blots of antibodies raised against cytochrome P-450con in rainbow trout displayed higher amounts of cytochrome P-450con with liver microsomes from sexually mature males than with sexually mature females. Cytochrome P-450con seems to be induced by cortisol and pregnenolone-16α-carbonitrile in juvenile rainbow trout liver. In addition, cytochrome P-450con is structurally related to a member of the glucocorticoid-inducible cytochrome P-450III family in rat and/or the major rat kidney form, cytochrome P-450k.
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.
Liver neoplasms were induced in medakas (Oryzias latipes) by the addition of diethylnitrosamine (DENA) to their aquarium water at levels of 15-135 ppm for 8 weeks. After 13 weeks, 21 to 32 fish had developed hepatomas. Medakas are useful for further studies because they are highly susceptible to the carcinogenic effect of DENA, and the time for tumor induction is relatively short. Histologic type differed in the lesions of different fish and also within individual tumors. Some were typical trabecular hepatomas, others were anaplastic hepatomas or cholangiomas, or mixtures of these. Electron microscopy revealed an extensive rough-surfaced endoplasmic reticulum in a lamellar pattern, many mitochondria, and several round lysosomes in tumor cells. A few fat droplets with occasional crystalline ghosts were sometimes in the cytoplasm. The Golgi apparatus was not conspicuous. Some cells had highly developed microvilli that showed differentiation toward structures resembling bile capillaries.
The histology of a spontaneously occurring neoplasm of the rat kidney conforming to a classification of nephroblastoma is described and compared with that of N-nitrosodimethylamine-induced renal mesenchymal tumors. This rat nephroblastoma was an encapsulated epitheloid neoplasm with a uniform histologic pattern. Clumps of densely crowded, hyperchromatic cells frequently associated with central, well-differentiated ducts were supported by a less cellular, interconnecting stroma of loose areolar or mature fibrous connective tissue. Neoplastic cells were organized into primitive, ill-defined tubular formations. The neoplastic cell component strongly resembled metanephrogenic blastema. In contrast, the renal mesenchymal tumor was nonencapsulated and consisted of a heterogeneous mixture of connective tissue elements including fibroblast-like spindle cells, smooth muscle, and embryonic mesenchyme that engulfed and sequestered preexisting renal tubules and glomeruli. The separate morphologic identities and apparently unrelated existence of rat nephroblastoma and renal mesenchymal tumor were stressed. The rat nephroblastoma morphologically resembled the malignant epithelial component of human Wilms' tumor, whereas rat renal mesenchymal tumor appeared to have counter-parts in the mesenchymal component of Wilms' tumor and in congenital mesoblastic nephroma (leiomyomatous hamartoma) of infancy. The histologic descriptions of previously recorded occurrences of spontaneous and experimentally induced rat neoplasma classified as nephroblastoma or its synonyms were reevaluated in comparison to the present case. In all but four instances, in which sufficient histologic detail was provided in previous reports, a consistent histologic pattern emerged for this neoplasm in the rat.
Liver cancer in rainbow trout was induced by exposure of fertile eggs to an aqueous, 0.5 ppm (microgram/ml) solution of aflatoxin B1 (AFB1) for 1 hour. Single treatments, given on alternate days during the embryonic period, produced a low cancer incidence (less than 20%) prior to formation of the embryonic liver on day 14, but a steadily increasing incidence from day 15 (31.7%) until day 23 (58.3%), in fish examined 1 year later. Treatment of trout eggs with [14C]AFB1 was used to quantitate the amount of AFB1 absorbed by the eggs. Twenty-one-day-old rainbow trout eggs absorbed approximately 30 ng of [14C]AFB1 during a 1-hour exposure to 0.5 ppm aqueous [14C]AFB1. After 1 day 85-90% of the [14C]AFB1 was either metabolized and excreted or leached from the egg. The residual [14C]AFB1 remained constant until hatching when an additional 50% was lost. Comparison of the amount of AFB1 absorbed by eggs with the amount of AFB1 consumed per fish during a 1-year feeding trial at 4 ppb in the diet indicates that the trout embryo is even more sensitive than juvenile trout to the carcinogenic properties of AFB1.
Four-week-old rainbow trout (Salmo gairdneri) were fed diets containing 0, 3, 50, 200, 400, and 800 ppm dimethylnitrosamine (DMN) for 52 weeks. At the end of 52 weeks, the fish were fed a control diet without DMN for an additional 26 weeks. Samples were taken at 26, 52, and 78 weeks to determine tumor incidence. A dose-related carcinogenic response was established from these results, and an equation was derived to relate the level of the carcinogen to the hepatocellular carcinoma incidence. From a published dose-response study that used outbred Porton rats, a second equation was derived for comparison. Rats and trout were approximately equal in their sensitivity to DMN carcinogenesis. The median lethal dose after ip injection of DMN was 1,770 mg/kg body weight in rainbow trout. Relative to the range of 15-50 mg/kg body weight reported for several mammalian species, trout were resistant to the acute toxicity of DMN.
Liver cancer appeared in domesticated rainbow trout in many fish hatcheries in the USA from 1957 to 1960. This unexpected disease outbreak reached epizootic proportions. Viruses, pesticides, and herbicides were suggested as possible causes, but at that time there was taking place a marked change in fish husbandry. Formerly, trout diets consisted of a 'wet' mixture of slaughterhouse by-products, with raw fish and horsemeat. Economic factors forced a change to a 'dry' pelleted food composed of a combination of fish, meat, and vegetable meals combined with a mixture of essential vitamins and minerals. By solvent extraction and thin-layer chromatography, Engebrecht et al. isolated from the toxic cottonseed meal a fluorescent substance and established by chemical tests and duckling assay that the compound was aflatoxin B1 (AFB). The same lesions were seen, however, with orally administered dimethylnitrosamine and cyclopropenoid fatty acids (CPFA), so it appears that the trout responds in a similar manner to different types of hepatic carcinogens, as does the rat. CPFA result in typical mature carcinomas, but there are additional preneoplastic nodular stages, the significance of which is unknown. The pathogenesis of CPFA-induced carcinogenesis needs to be studied in much greater detail. This brief review indicates that there is much which is presently not known about the pathogenesis of rainbow trout liver tumors. In particular, there is a need to investigate the histochemical characteristics of trout livers early in the carcinogenic process, to manipulate carcinogen application in an attempt to induce early morphologically recognizable preneoplastic lesions, to experiment with a greater spectrum of recognized liver carcinogens, and to further develop the trout egg-carcinogen bath method as a tool to understand the dynamics of one-dose carcinogenesis. ( 82 references).
Weekly s.c. injections of equitoxic doses of 2-hydroxy-propyl-n-propylnitrosamine, 2-oxopropyl-n-propylnitrosamine, and methyl-n-propylnitrosamine, assumed metabolites of di-n-propylnitrosamine by beta oxidation, induced low incidences of pancreatic duct adenomas in Syrian golden hamsters. Di-n-propylnitrosamine did not. Application of 2,2'-dihydroxydi-n-propylnitrosamine, another postulated intermediate of di-n-propylnitrosamine, led to development of various types of pancreatic duct adenomas and ductal carcinomas in high percentages of hamsters. In addition, a few acinar-cell carcinomas were found. The morphology of these neoplasms, their latencies, and their distribution in the different segments of the pancreas are described.
The bioactivation of 7-hydroxymethyl-12-methylbenz[a]anthracene (HMBA) to an electrophilic sulfuric acid ester metabolite has been shown to be catalyzed by rat liver bile acid sulfotransferase I (BAST I). The sulfation and activation of HMBA by BAST I was determined by the ability of sulfated HMBA to form DNA adducts. The BAST I was also shown to react with rabbit anti-human dehydroepiandrosterone sulfotransferase antisera and to represent a major form of hydroxysteroid/bile acid sulfotransferase in female rat liver cytosol. Higher levels of BAST I activity and immunoreactivity as well as HMBA-DNA adduct formation were detected in female rat liver cytosol than in male rat liver cytosol. The bioactivation of HMBA by pure BAST I was dependent on the presence of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in the reaction and was inhibited by dehydroepiandrosterone, a physiological substrate for BAST I. Glutathione, a cellular nucleophile with important protective properties, decreased DNA adduct formation in the HMBA sulfation reaction in the absence of glutathione S-transferase activity. These results indicate the usefulness of BAST I to investigate the sulfation and activation of HMBA and probably other hydroxymethylated polyaromatic hydrocarbons to electrophilic and mutagenic metabolites under defined reaction conditions.
Dehydroepiandrosterone (DHEA), a C19 adrenal steroid hormone, induces peroxisome proliferation in liver cells and is hepatocarcinogenic in the rat. The present study deals with the phenotypic properties of DHEA-induced liver lesions. A majority of the altered areas (80-87%), neoplastic nodules (> 94%) and hepatocellular carcinomas (HCC, 80-100%) lacked the marker enzymes gamma-glutamyltranspeptidase and placental form of glutathione S-transferase (GSTP). Northern blot analysis of HCC from 4 rats revealed no detectable GSTP mRNA. These HCC, however, showed a marked decrease in the staining of glucose-6-phosphatase and adenosine triphosphatase. These results indicate that the phenotypic properties of liver tumors induced by DHEA and amphipathic carboxylate peroxisome proliferators are similar.
Much evidence supports the view that the rate of conjugation of glutathione (GSH) with aflatoxin B1 (AFB1) exo-epoxide is an important factor in determining the species variation in risk to aflatoxins and that induction of GSH S-transferases can yield a significant protective effect. An assay has been developed in which the enzymatic formation of the conjugates of GSH and AFB1 exo-epoxide and the recently described AFB1 endo-epoxide is measured directly. 1H NMR spectra are reported for both the AFB1 exo- and endo-epoxide-GSH conjugates. Structural assignments were made by comparison with AFB1 exo- and endo-epoxide-ethanethiol conjugates, for which nuclear Overhauser effects were measured to establish relative configurations. The endo-epoxide was found to be a good substrate for GSH conjugate formation in rat liver cytosol while mouse liver cytosol conjugated the exo-epoxide almost exclusively. Human liver cytosol conjugated both epoxide isomers to much lower extents than did cytosols prepared from rats or mice. Purified rat GSH S-transferases catalyzed the formation of the AFB1 exo-epoxide-GSH conjugate in the order 1-1 approximately 4-4 approximately 3-3 greater than 2-2 greater than 4-6 (7-7 and 8-8 did not form the exo-epoxide-GSH conjugate at levels above the nonenzymatic rate). The only rat GSH S-transferases that conjugated the endo-epoxide were 4-4 and 4-6, with 4-4 being the more active.(ABSTRACT TRUNCATED AT 250 WORDS)
The tumor-enhancing effect of hydrogen peroxide (H2O2) in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-initiated rainbow trout hepatocarcinogenesis was investigated and correlated with the levels of the mutagenic DNA adduct 8-hydroxy-2'-deoxyguanosine (oh8dG). In addition, the protective role of vitamin E was examined in relation to tumor enhancement and oh8dG levels in liver DNA. Trout were fed diets containing two levels of vitamin E (1000 or 20 mg/kg wet wt), each of which were made up to contain three levels of H2O2 (0, 600 or 3000 p.p.m.). Dietary vitamin E levels had no significant effect on tumor incidence or levels of oh8dG in liver DNA. On the other hand, dietary H2O2 enhanced liver tumors in a dose-dependent manner. Liver tumor incidence correlated significantly with the mean level of liver DNA oh8dG content (r = 0.87). We conclude that the H2O2 tumor-enhancing effect coincides with higher levels of oh8dG in the trout liver genome. Thus, rainbow trout may be a useful model for the study of the relationship of oh8dG levels in vivo to enhancement or promotion of carcinogenesis and its modulation by dietary enhancers and inhibitors of oxidative stress.
The complementary DNAs of rat glutathione S-transferase (GST, EC 2.5.1.18) Yc1 and of mouse Yc were expressed from a prokaryotic expression vector in E. coli. The purified proteins were analyzed for their activity toward aflatoxin B1-8,9-epoxide (AFBO), the reactive intermediate of the fungal mycotoxin aflatoxin B1 (AFB). The mouse Yc isozyme had about 50-fold higher conjugating activity toward AFBO than the rat Yc1 isozyme (144 nmol/mg/min versus 3.3 nmol/mg/min). The rat Yc1 isozyme had specific activities toward 1-chloro-2,4-dinitrobenzene, cumene hydroperoxide and ethacrynic acid of 10.7, 0.98 and 0.92 mumol/mg/min, respectively, whereas the mouse Yc isozyme had specific activities of 5.7, 2.1 and 0.1 mumol/mg/min for these substrates, respectively. These data provide further support for the hypothesis that the constitutive presence of the alpha class GST Yc isozyme in mouse liver protects mice from the hepatocarcinogenic effects of aflatoxin B1.
Cytochrome P-450 dependent monooxygenases play a dual role for xenobiotic metabolism. On one hand they initiate the primary rate limiting step for the elimination of a bulk of drugs and organic chemicals. On the other hand they catalyze the formation of toxic metabolites from chemical carcinogens and many other toxic chemicals. Numerous studies have shown that their activity in animals is subject to the influence of various modifying factors, such as strain, species, sex, age, diurnal rhythm and the effect of enzyme inducers. Less is known about the influence of these factors on human cytochrome P-450 enzymes. Here we report the results of an extended study on human liver cytochrome P-450 performed with liver biopsies of 178 individuals taken for diagnostic purposes. The enzymatic activity was determined by the aldrin epoxidase assay indicating a variety of enzymes inducible by phenobarbital and by glucocorticoid and androgenic hormones. The frequency histogram of individual aldrin epoxidase activities showed a unimodal distribution and a variation factor of 100 between maximal and minimal activity. Individuals with severe liver diseases, such as cirrhosis and fatty liver, exhibited a 50% loss of enzyme activity. Age and sex did not significantly influence the enzyme activity. No significant correlation was observable between the rate of aldrin epoxidation and debrisoquine 4-hydroxylation, a prototype of a genetically controlled cytochrome P-450 reaction. We assume that the broad interindividual variation of epoxidase activities is more likely due to the influence of exogenous and endogenous inducers rather than to a genetic polymorphism.(ABSTRACT TRUNCATED AT 250 WORDS)
We compared the profile of ras gene mutations in spontaneous CD-1 mouse liver tumors with that found in liver tumors that were induced by a single i.p. injection of either 7,12-dimethylbenz(a)anthracene (DMBA), 4-aminoazobenzene, N-hydroxy-2-acetylaminofluorene, or N-nitrosodiethylamine. By direct sequencing of polymerase chain reaction-amplified tumor DNA, the carcinogen-induced tumors were found to have much higher frequencies of ras gene activation than spontaneous tumors. Furthermore, each carcinogen caused specific types of ras mutations not detected in spontaneous tumors, including several novel mutations not previously associated with either the carcinogen or mouse hepatocarcinogenesis. For example, the model compound DMBA is known to cause predominantly A to T transversions in Ha-ras codon 61 in mouse skin and mammary tumors, consistent with the ability of DMBA to form bulky adducts with adenosine. Our results demonstrate that the predominant mutation caused by DMBA in mouse liver tumors is a G to C transversion in Ki-ras codon 13 (DMBA is also known to form guanosine adducts), illustrating the influence of both chemical- and tissue-specific factors in determining the type of ras gene mutations in a tumor. 4-Aminoazobenzene and N-hydroxy-2-acetylaminofluorene also caused the Ki-ras codon 13 mutation. In addition, we found that N-nitrosodiethylamine, 4-aminoazobenzene, and N-hydroxy-2-acetylaminofluorene all caused G to T transversions in the N-ras gene (codons 12 or 13). This is the first demonstration of N-ras mutations in mouse liver tumors, establishing a role for the N-ras gene in mouse liver carcinogenesis. Finally, comparison of the ras mutations detected in the direct tumor analysis with those detected after NIH3T3 cell transfection indicates that spontaneous ras mutations (in Ha-ras codon 61) are often present in only a small fraction of the tumor cells, raising the possibility that they may sometimes occur as a late event in CD-1 mouse hepatocarcinogenesis.
The DNA adducts of 7,12-dimethylbenz[a]anthracene (DMBA) previously identified in vitro and in vivo are stable adducts formed by reaction of the bay-region diol epoxides of DMBA with dG and dA. In this paper we report identification of several new DMBA-DNA adducts formed by one-electron oxidation, including two adducts lost from DNA by depurination, DMBA bound at the 12-methyl to the N-7 of adenine (Ade) or guanine (Gua) [7-methylbenz[a]anthracene (MBA-12-CH2-N7Ade or 7-MBA-12-CH2-N7Gua, respectively]. The in vitro systems used to study DNA adduct formation were DMBA activated by horseradish peroxidase or 3-methyl-cholanthrene-induced rat liver microsomes. The biologically-formed depurination adducts were identified by high-pressure liquid chromatography and by fluorescence line narrowing spectroscopy. Stable DMBA-DNA adducts were analyzed by the 32P-postlabeling method. Quantitation of DMBA-DNA adducts formed by microsomes showed about 99% as depurination adducts: 7-MBA-12-CH2-N7Ade (82%) and 7-MBA-12-CH2-N7Gua (17%). Stable adducts (1.4% of total) included one adduct spot that may contain adduct(s) formed from the diol epoxide (0.2%) and unidentified adducts (1.2%). Activation of DMBA by horseradish peroxidase afforded 56% of stable unidentified adducts and 44% of depurination adducts, with 36% of 7-MBA-12-CH2-N7Ade and 8% of 7-MBA-12-CH2-N7Gua. Adducts containing the bond to the DNA base at the 7-CH3 group of DMBA were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)
The suspect human hepatocarcinogen aflatoxin B1 (AFB1) is a well-known potent initiator of hepatic tumors in rainbow trout (Oncorhynchus mykiss). Both hepatocellular carcinomas and mixed hepatocellular/cholangiocellular carcinomas are induced by AFB1 in trout, with the mixed form predominating. We previously isolated two c-ras genes from trout liver cDNA, and in the present study we analyzed DNA from 14 AFB1-induced trout liver tumors for point mutations in exon 1 of both genes. Using the polymerase chain reaction (PCR) and oligonucleotide hybridization methods, a high proportion (10/14) of the AFB1-initiated tumor DNAs showed evidence of activating point mutations in the trout c-Ki-ras gene. Of the 10 mutant ras genotypes, seven were codon 12 GGA----GTA transversions, two were codon 13 GGT----GTT transversions, and one was codon 12 GGA----AGA transition. Nucleotide sequence analysis of cloned PCR products from four of these tumor DNAs provided definitive evidence for two codon 12 GGA----GTA mutations, one codon 12 GGA----AGA mutation, and one codon 13 GGT----GTT mutation, in complete agreement with the oligonucleotide hybridization results. No mutations were detected in exon 1 of a second trout ras gene also expressed in liver, nor in DNA from control livers. This is the first report of experimentally induced ras gene point mutations in a lower vertebrate fish model. The results indicate that the hepatocarcinogen AFB1 induces c-Ki-ras gene mutations in trout similar to those in rat liver tumors.
A variety of neoplasms and nonneoplastic hepatic lesions have been noted in winter flounder, Pseudopleuronectes americanus, from Boston Harbor, Massachusetts. Inflammatory lesions include cholangiitis, pericholangiitis, pericholangial fibrosis, hepatitis, and pancreatitis. Necrotic lesions consist essentially of focal coagulative necrosis and a distinctive vacuolated cell lesion of the hepatic parenchyma. The most conspicuous and numerous proliferative lesion is macrophage aggregate hyperplasia and hypertrophy. Preneoplastic lesions include principally basophilic foci of cellular alteration and hepatocellular adenoma. Carcinomas consist of several morphologic varieties: hepatocarcinoma, cholangiocarcinoma, and anaplastic adenocarcinoma. The pathogenesis of the lesions observed is discussed with respect to anthropogenically introduced chemical contaminants and the resistant hepatocyte model of hepatocarcinogenesis. This study, and others of bottom-living food fish with enzootic neoplastic disease, warrants further evaluation, particularly with respect to possible bioaccumulation of chemical contaminants in edible tissues.
The fine structure of hepatocellular neoplasms from aflatoxin B1 (AFB1)-initiated rainbow trout was studied by transmission electron microscopy. Large, usually uniform hepatic nuclei, large nucleoli, abundant, dilated rough-surfaced endoplasmic reticulum, and reduced glycogen storage were common findings in both hepatocellular adenomas and hepatocellular carcinomas. In addition, the presence of poorly developed microvilli in the space of Disse and in bile canaliculi, the occurrence of few or no bile preductule cells and a striking increase in the size and number of intercellular spaces characterized hepatocellular carcinomas. The three latter characteristics of hepatocellular carcinomas suggest loss of inter-relationships between hepatocytes and the microvascular system (sinusoids), between hepatocytes and the biliary system, and between individual hepatocytes, respectively. With respect to these parameters, adenomas were more similar to normal liver than to carcinomas.
The DNA sequences around codons 12, 13, and 61 of the ras gene were analyzed by polymerase chain reaction and direct sequencing in 18 intrahepatic cholangiocarcinomas. The ras gene mutations were found in 9 of 18 (50%): 6 in K-ras codon 12, 1 in K-ras codon 13, 1 in K-ras codon 61, and 1 in N-ras codon 12. The incidence of mutations was higher in the hilar type of intrahepatic cholangiocarcinomas, especially when these tumors were large. The incidence and spectrum of the mutations were almost the same as those reported in colon cancers, possibly indicating similar etiologic agent(s) in the carcinogenesis of both cancers.
Although a bay-region dihydrodiolepoxide metabolite has been considered as a principal ultimate electrophilic and carcinogenic form of 7,12-dimethylbenz[a]anthracene (DMBA), other reactive metabolites might also play a role in the activation of this hydrocarbon in vivo. Earlier studies suggested the hydroxylation of a meso-anthracenic methyl group with subsequent formation of a benzylic ester bearing a good leaving group (e.g. sulfate) as a metabolic activation pathway for DMBA. In support of this hypothesis, the formation of an electrophilic and mutagenic sulfuric acid ester of 7-hydroxymethyl-12-methylbenz[a]anthracene (HMBA) by rat liver cytosolic sulfotransferase activity has previously been demonstrated, but no data have been reported on the carcinogenicity of this reactive ester. In the present study, we compared the carcinogenicity of chemically synthesized sodium 7-sulfooxymethyl-12-methylbenz[a]anthracene (SMBA) with that of the parent methyl and hydroxymethyl hydrocarbons. For this purpose, tests were made in several animal tumor models: induction of hepatomas in male B6C3F1 mice, lung adenoma induction in A/J mice, initiation of mouse skin tumors, development of sarcomas in rats at the injection sites, and initiation of preneoplastic enzyme-altered foci in rat liver. Data from all of these studies indicate that SMBA is not more carcinogenic than DMBA or HMBA. In addition, the carcinogenic activity of HMBA was not altered by dehydroepiandrosterone, a strong inhibitor of sulfotransferase activity for HMBA. DMBA produced only a low level of hepatic benzylic DNA adducts in rats when a relatively high dose was administered. These adducts constitute <5% of total DMBA residues bound to hepatic DNA. The rest of the adducts appear to be associated with other electrophilic intermediates including the dihydrodiol epoxide metabolites. Based on the results of our present study, it is unlikely that DMBA exerts its carcinogenic activity via metabolic activation to SMBA.
Studies were undertaken to determine the immunochemical relationship between constitutive trout cytochrome P450s and mammalian cytochrome P450IIIA enzymes. Polyclonal antibodies (IgG) generated against trout P450 LMC5 reacted strongly with P450IIIA1 in dexamethasone-induced rat liver microsomes and with P450IIIA4 in human liver microsomes in immunoblots. In contrast, rabbit anti-P450 LMC1 IgG did not recognize these proteins in rat and human liver microsomes. Reciprocal immunoblots using anti-rat P450IIIA1 showed that this antibody does not recognize trout P450 LMC1 or LMC5. However, anti-human P450IIIA4 IgG was found to cross react strongly with P450 LMC1 and LMC5. Progesterone 6 beta-hydroxylase activity of trout liver microsomes, a reaction catalyzed by P450 LMC5, was markedly inhibited by anti-P450IIIA4 and by gestodene, a mechanism-based inactivator of P450IIIA4. These results provide evidence for a close structural similarity between trout P450 LMC5 and human P450IIIA4.
Members of the ras gene family have been studied in a variety of species. Two ras genes expressed in the normal liver of rainbow trout, ras-1 and ras-2, as well as a portion of a genomic ras-1 allele, are described for the first time in this report. Over 500 bp of trout ras-1 and at least 300 bp of trout ras-2 genes expressed in normal liver have been sequenced; DNA homology to the human ras genes ranges from 76.8% to 87.1%. The base changes resulting from over 400 million years of evolutionary divergence between the species were primarily silent, with few changes in protein sequence. The partial DNA sequence of the genomic ras-1 allele has 86.8% homology to the first two exons of human c-Ha-ras, and its intron has several conserved sequences characteristic of vertebrate intron-exon junctions. However, the predicted amino acid sequence of trout ras-1 differs at only one of the first 172 amino acid residues from human c-Ki-ras with the alternate exon 4b. Since trout ras-1 differs at 17 and 18 residues from the human c-N-ras and c-Ha-ras proteins, respectively, over this region, we conclude that trout ras-1 is a c-Ki-ras gene. The highly conserved nature of this gene suggests that the ras p21 protein has identical functions in normal and neoplastic cells among higher and lower vertebrates.
High levels of polynuclear aromatic hydrocarbon (PAH) carcinogens commonly occur in aquatic systems where neoplasms arise in fish and other animals. Enzymes that transform PAHs can act in initiating these diseases and can indicate the contamination of fish by carcinogens and other pollutants. Cytochrome P-450 has similar roles in activating PAH carcinogens in fish and mammalian species. PAHs and many chlorinated hydrocarbons, e.g., polychlorinated biphenyls (PCBs) induce a form of cytochrome P-450 in fish that is the primary catalyst of PAH metabolism. The induction of this P-450 in fish can accelerate the disposition of hydrocarbons, but can also enhance the formation of carcinogenic derivatives of PAHs. Invertebrates have lower rates of PAH metabolism than fish. These rates are not obviously inducible by exposure to PAHs or PCBs. The lower rates of foreign compound metabolism contribute to higher pollutant residue levels in bivalve mollusks (clams, mussels, etc.) than in fish and may limit the involvement of some procarcinogens (requiring activation) in disease processes in invertebrates. The induction of P-450 forms can indicate the exposure of fish to PAHs, PCBs, and other toxic compounds. This is not restricted to carcinogens. Environmental induction has been detected in fish from contaminated areas by use of catalytic assay, antibodies to fish P-450, and cDNA probes that hybridize with P-450 messenger RNA. Application of these methods can provide sensitive biological monitoring tools that can detect environmental contamination of fish by some carcinogens and tumor promoters.(ABSTRACT TRUNCATED AT 250 WORDS)
Dibenzo[a,l]pyrene (DB[a,l]P) is one of the most potent carcinogens ever tested in mouse skin and rat mammary gland. DB[a,l]P is present in cigarette smoke and, presumably, in other environmental pollutants. Metabolism and mutagenicity studies of this compound compared to the weak carcinogen dibenzo[a,e]pyrene (DB[a,e]P) can provide preliminary evidence on its mechanism of carcinogenesis. The mutagenicity of DB[a,l]P, DB[a,e]P, and benzo[a]pyrene (BP) was compared in the Ames assay with Aroclor-induced rat liver S-9. BP was the strongest mutagen. In strain TA100, DB[a,l]P and DB[a,e]P were marginally mutagenic. In strain TA98 both compounds were mutagenic, and DB[a,l]P induced more than twice as many revertants as DB[a,e]P. The mutagenicity of DB[a,l]P does not correlate with its carcinogenicity, since DB[a,l]P is a much stronger carcinogen, but a much weaker mutagen, than BP. The NADPH-supported metabolism of DB[a,e]P and DB[a,l]P was conducted with uninduced and 3-methylcholanthrene-induced rat liver microsomes. Metabolites were analyzed by reverse-phase HPLC and identified by NMR, UV, and mass spectrometry. Uninduced microsomes produced only traces of metabolites with either compound. The major metabolites of DB[a,l]P with induced microsomes were DB[a,l]P 8,9-dihydrodiol, DB[a,l]P 11,12-dihydrodiol, 7-hydroxyDB[a,l]P, and a DB[a,l]P dione. The metabolites of DB[a,e]P with induced microsomes were DB[a,e]P 3,4-dihydrodiol, 3-hydroxyDB[a,e]P, 7-hydroxyDB[a,e]P, and 9-hydroxyDB[a,e]P. Some of these metabolites are very useful in assessing possible pathways of activation in the initiation of cancer.