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Toxicity of 4-chloroaniline in early life-stages of zebrafish (Brachydanio rerio): I. Cytopathology of liver and kidney after microinjection
Abstract
In addition to survival and hatching parameters, cytological alterations in liver and kidney of 4- and 6-d old zebrafish larvae (Brachydanio rerio) following single microinjection of fertilized eggs at the germ-ring stage with 5, 12.5, and 25 ng 4-chloroaniline/egg were investigated by means of electron microscopy. Whereas survival remained unaffected, microinjection with 4-chloroaniline disturbed hatching of larvae. Hatching was delayed by microinjection of 12.5 ng 4-chloroaniline/egg and above when compared to controls. Cytological investigations revealed ultrastructural changes in both liver and kidney in a dose- and time-dependent fashion. In the liver, major cytopathological changes included fenestration, fragmentation, and vesiculation of the rough endoplasmic reticulum, proliferation of atypical mitochondria, and atypical lysosomes. Furthermore, myelin whorls, lipid inclusions, and cholesterol crystals were increased, whereas glycogen stores were reduced. Renal tubular cells displayed altered brush borders, proliferation of nucleoli, atypical mitochondria, fenestrated, fragmented, and vesiculated RER cisternae, as well as giant lysosomes. Most of these effects indicate cellular dysfunction (e.g., disturbance of lipid metabolism in the liver), whereas others illustrate general cellular stress-responses to chemical aggression. Comparisons of results with those of previous studies based on conventional fish exposure prove the suitability and sensitivity of microinjection bioassays with zebrafish eggs as an alternative to conventional early life-stage tests.
... According to the dates of these publications, we can say that the evaluation of mitochondrial alterations using D. rerio has been recent. One of the first studies to be published on mitotoxicity using zebrafish as a model organism was by Oulmi and Braunback [113], who at the time were microinjecting D. rerio embryos to evaluate an organochlorine compound (4-chloroaniline) used to make dyes, insecticides, and various industrial products. In this work, for the toxicological analysis, the researchers evaluated the size, shape, and function of the mitochondria in relation to the quantity of other organelles in the liver and kidney, concluding that, as well as the zebrafish being a suitable model for microinjection tests, it is also suitable in terms of histological and cytological methods for diagnosing the potential dangers of environmental chemical products [113]. ...
... One of the first studies to be published on mitotoxicity using zebrafish as a model organism was by Oulmi and Braunback [113], who at the time were microinjecting D. rerio embryos to evaluate an organochlorine compound (4-chloroaniline) used to make dyes, insecticides, and various industrial products. In this work, for the toxicological analysis, the researchers evaluated the size, shape, and function of the mitochondria in relation to the quantity of other organelles in the liver and kidney, concluding that, as well as the zebrafish being a suitable model for microinjection tests, it is also suitable in terms of histological and cytological methods for diagnosing the potential dangers of environmental chemical products [113]. ...
This chapter provides an overview of the zebrafish (Danio rerio) as a model organism for studies of mitochondrial dysfunction. Zebrafish possess a genetic similarity with humans and have conserved mitochondrial genomes, rendering them a valuable research tool for examining the intricate mechanisms that govern mitochondrial processes at diverse developmental stages. The chapter explores several methods for evaluating mitochondrial health and function. Examples include in vitro cell culture and in vivo analysis in embryos, larvae, and adults. The chapter discusses the use of zebrafish models in toxicological research to investigate mitochondrial reactions to environmental stressors and xenobiotics. The importance of implementing standardized protocols, validating marker, integrating different omics data, and using in vivo and in vitro approaches to advance mitochondrial research will be highlighted. In summary, zebrafish are suitable for analyzing both mitochondrial function and dysfunction, as well as their impact on human health.
... However, the study of fish kidney is still an undervalued matter, particularly with the application of quantitative methods. To our knowledge, only a few semiquantitative studies were performed in this organ, and they were always associated with toxicological settings~Rojik et al., 1983;Oulmi et al., 1995aOulmi et al., , 1995bOulmi & Braunbeck, 1996;Kamunde et al., 1997!. Therefore, the goal of this study was to characterize qualitatively and quantitatively the trunk kidney of brown trout, providing baseline data for further descriptive or experimental studies. ...
... Fish renal morphology has been studied for a long time, but the vast heterogeneity of this organ among fish species makes the available literature still quite insufficient for an in depth picture. Studies quantitatively characterizing the renal structure are even more rare~Kamunde et al., 1997! and mostly regarding toxicological studies~Oulmi et al. , 1995a, 1995bOulmi & Braunbeck, 1996;Weber et al., 2003;Triebskorn et al., 2004!. Thus, this work aimed to provide a qualitative characterization of the several components from brown trout kidney and also a quantitative analysis, with stereological methods, for providing baseline data to support morphofunctional correlations, and also to determine whether changes existed in the renal components during the male and female reproductive cycle. ...
The large variability in kidney morphology among fish makes it difficult to build a "universal" model on its function and structure. Therefore, a morphological study of brown trout trunk kidney was performed, considering potential seasonal and sex effects. Three-year-old specimens of both sexes were collected at four stages of their reproductive cycle. Kidney was processed for light and electron microscopy. The relative volumes of renal components, such as renal corpuscles and different nephron tubules, were estimated by stereological methods. Qualitatively, the general nephron structure of brown trout was similar to that described for other glomerular teleost species. Quantitatively, however, differences in the relative volume of some renal components were detected between sexes and among seasons. Particularly, highest values of vacuolized tubules and new growing tubules were observed after spawning, being more relevant in females. Despite seasonal changes, more linear correlations were found between those parameters and the reno-somatic index than the gonado-somatic index. Thus, we verified that some brown trout renal components undergo sex dependent seasonal variations, suggesting a morphological adaptation of the components to accomplish physiological needs. These findings constitute a baseline for launching studies to know which factors govern the morphological variations and their functional consequences.
... Zebrafish have increasingly gained attention as a model organism for biomedical research such as organelle biology research [42,43], developmental disorders research [44,45], mental disorders research [46,47], and communication between the brain and organs research [48]. Zebrafish have been shown [49] to possess the potential of a model organism for drug discovery, exploring mechanisms of drug action in vivo, and preclinical drug repurposing: hence its emergence as a leading model organism in human disease. ...
The unrelenting increase in the incidence of type 2 diabetes (T2D) necessitates the urgent need for effective animal models to mimic its pathophysiology. Zebrafish possess human-like metabolic traits and share significant genetic similarities, making them valuable candidates for studying metabolic disorders, including T2D. This review emphasizes the critical role of animal models in diabetes research, especially focusing on zebrafish as an alternative model organism. Different approaches to a non-genetic model of T2D in zebrafish, such as the glucose solution, diet-induced, chemical-induced, and combined diet-induced and glucose solution methods, with an emphasis on model validation using indicators of T2D, were highlighted. However, a significant drawback lies in the validation of these models. Some of these models have not extensively demonstrated persistent hyperglycemia or response to insulin resistance and glucose tolerance tests, depicted the morphology of the pancreatic β-cell, or showed their response to antidiabetic drugs. These tools are crucial in T2D pathology. Future research on non-genetic models of T2D in zebrafish must extensively focus on validating the metabolic deficits existing in the model with the same metabolic defects in humans and improve on the existing models for a better understanding of the molecular mechanisms underlying T2D and exploring potential therapeutic interventions.
... Zebrafish have served as an excellent model system to assess in vivo toxicity in response to treatment of a chemical of interest, and numerous studies have illustrated metabolic changes related to mitochondrial function upon chemical treatment [55][56][57] . After an initial study of mitochondrial activity and distribution in zebrafish oocytes reported in 1980, many reports regarding the mitochondrial genome and functional homologs of mitochondrial proteins in zebrafish were published in the late 1990s and early 2000s. ...
Zebrafish have several advantages compared to other vertebrate models used in modeling human diseases, particularly for large-scale genetic mutant and therapeutic compound screenings, and other biomedical research applications. With the impactful developments of CRISPR and next-generation sequencing technology, disease modeling in zebrafish is accelerating the understanding of the molecular mechanisms of human genetic diseases. These efforts are fundamental for the future of precision medicine because they provide new diagnostic and therapeutic solutions. This review focuses on zebrafish disease models for biomedical research, mainly in developmental disorders, mental disorders, and metabolic diseases.
... Hematotoxicity may also be a contributing factor to the splenotoxicity induced by anilines. Toxicity studies in zebrafish demonstrate that 4-chloroaniline is embryotoxic, impacting liver, kidney and gills (Burkhardt-Holm et al., 1999;Oulmi et al., 1996). There is only limited toxicological data for this environmental contaminant. ...
There is an increasing demand by regulators, pharmaceutical manufacturers and industry for reliable and ethically acceptable model systems to assess and predict the toxicity of pharmaceutical products, chemicals, and waste. In particular, developmental toxicity of compounds has largely been ignored in the past raising massive concerns. While cell lines have great merits as test systems, they do not reflect the complexity of a developing embryo. Zebrafish and their embryos have been used in toxicity assays in the past and have a great potential for studying reproductive toxicity and the molecular basis of developmental toxicity. Toxicogenomics is a powerful tool for compound classification based on mechanistic studies, which could eventually lead to the prediction of the toxicity of novel compounds. I have carried out here a systematic toxicogenomic study of zebrafish embryos with the aim to develop this system further as a model for molecular developmental toxicity studies. Questions such as stage- and compound specificity were addressed. The toxicogenomic responses to a series of 6 test compounds were highly stage-dependent. Moreover, exposure of late embryonic stages between 96 and 120 hours post-fertilisation induced transcriptional profiles that are characteristic for specific compounds. This leads to the identification of 199 genes that are induced by at least one of the 11 compounds including a number of signature genes that appear specific for individual compounds or compound groups within the set of chemicals investigated. Moreover, I have tested whether one can observe toxicogenomic responses in the absence of apparent morphological effects. In several instances robust gene responses were measured at concentrations that did not have obvious morphological effects. This raises hopes that toxicogenomic studies in the zebrafish embryo could be used to predict chronic effects of low level exposure. In situ hybridisation of a number of selected genes showed that the responses can be highly tissue restricted indicating that the whole mount protocol developed here is highly sensitive. In conclusion, this toxicogenomic analysis, demonstrated that zebrafish embryos may be suitable to study the transcriptional responses to toxicants with high specificity and more sensitivity. The zebrafish may thus be developed into a system that will not only help to elucidate the molecular mechanisms of toxicity but may be also useful to predict the developmental toxicity of novel chemicals by comparison of toxicogenomic profiles.
... Additionally, our results suggest that MXC (or its derivatives) can interact with PPAR and/or PPRE in zebrafish since MXC exposure provoked increased peroxisomal density values and induced activity of AOX. A modest peroxisomal proliferation has previously been observed in liver of 4 –6 day old larvae of zebrafish after injection of 4-chloroaniline (Oulmi and Braunbeck, 1996) but not in adult female zebrafish exposed to the same compound (Braunbeck et al., 1990). Peroxisome proliferation has been reported in different fish species exposed to other organochlorine pesticides such as endosulfan or dieldrin (Arnold et al., 1995; Pedrajas et al., 1996). ...
Environmental estrogenic compounds or xenoestrogens can mimic natural estrogens and cause a variety of adverse effects on aquatic wildlife. The purpose of the present work was to investigate if xenoestrogens are able to cause proliferation of liver peroxisomes using zebrafish (Danio rerio) as a model. Adult male zebrafish were exposed for 15 days to 17beta-estradiol (E2) and the xenoestrogens dibutylphthalate (DBP), methoxychlor (MXC), 4-tert-octylphenol (OP) and 17alpha-ethynylestradiol (EE2). All five tested compounds caused significant proliferation of liver peroxisomes (p < 0.05) as indicated by increased peroxisomal surface and numerical densities and elevated activities of the peroxisomal beta-oxidation enzyme acyl-CoA oxidase (AOX). In the case of DBP, MXC and E2, positive significant correlations between peroxisomal density parameters and AOX were found. The treatments did not produce gross alterations in testis histology, but spermatogenic cell proliferation was disturbed in E2 and EE2-treated groups and vitellogenin levels increased significantly in fish exposed to MXC, OP, EE2 and E2 with respect to controls. Furthermore, a significant correlation between vitellogenin levels and AOX activity was found for MXC, OP and EE2 treatments, suggesting that for the latter xenoestrogens early estrogenic effects are associated with liver peroxisome proliferation. No such association occurred with typical peroxisome proliferators such as DBP.
... The receptor name PPAR reflects the fact that its isoforms are activated by substances that increase the number and size of peroxisomes in the organism, which was first shown in rodents (Issemann and Green, 1990). Peroxisomes in teleost fish are known to proliferate after treatment with certain pesticides, phthalates, alkylphenols, and estrogens Cajaraville, 2000, 2005;Oulmi and Braunbeck, 1996). Three main PPAR subtypes have been identified in several mammals, amphibians and teleost fish: PPARa, PPARb and PPARc (Andersen et al., 2000;Boukouvala et al., 2004;Escriva et al., 1997;Ibabe et al., 2005;Leaver et al., 2005;Mano et al., 2000;Schoonjans et al., 1996). ...
Aromatic nitro and amino compounds have various commercial, governmental, and public applications. Their uses include manufacturing of dyes, pesticides, drugs, and explosives. These substances are typically considered high‐nitrogen compounds and as such share many of the same chemical and biological properties. Many have amines and/or nitrate groups on the ring and because of this exhibit properties where they are not highly water or lipid soluble and many tend to form crystals. When released to the environment, nitrate‐containing moieties are often reduced to amines by bacteria in wet soils and sediments. Acute effects typically target the nervous system; however, sublethal effects from repetitive exposures include anemia, methemoglobinemia, and often male reproductive effects. Cyanosis has been described from relatively high repetitive exposures. Oral exposures can affect the liver. Many are absorbed through the skin where dermal exposures can significantly contribute to systemic dose. Skin sensitization and irritation have been observed from exposure to some compounds. Many are eliminated via the urine; however, conjugated metabolites can be eliminated via the feces. Nitroso metabolites are suspected as having influence in observation of cancer, particularly in the bladder. Other similar nitramine compounds are also briefly described.
Emerging bio-contaminants such as viruses have affected health and environment settings of every country. Viruses are the minuscule entities resulting in severe contagious diseases like SARS, MERS, Ebola, and avian influenza. Recent epidemic like the SARS-CoV-2, the virus has undergone mutations strengthen them and allowing to escape from the remedies. Comprehensive knowledge of viruses is essential for the development of targeted therapeutic and vaccination treatments. Animal models mimicking human biology like non-human primates, rats, mice, and rabbits offer competitive advantage to assess risk of viral infections, chemical toxins, nanoparticles, and microbes. However, their economic maintenance has always been an issue. Furthermore, the redundancy of experimental results due to aforementioned aspects is also in examine. Hence, exploration for the alternative animal models is crucial for risk assessments. The current review examines zebrafish traits and explores the possibilities to monitor emerging bio-contaminants. Additionally, a comprehensive picture of the bio contaminant and virus particle invasion and abatement mechanisms in zebrafish and human cells is presented. Moreover, a zebrafish model to investigate the emerging viruses such as coronaviridae and poxviridae has been suggested.
Because of essential role in homeostasis of the body fluid and excretion of wastes, kidney damage can lead to severe impacts on health and survival of humans. For most chemicals, nephrotoxic potentials and associated mechanisms are unclear. Hence, fast and sensitive screening measures for nephrotoxic chemicals are required. In this study, the utility of zebrafish (Danio rerio) was evaluated for the investigation of chemical-induced kidney toxicity and associated modes of toxicity, based on the literature review. Zebrafish has a well-understood biology, and many overlapping physiological characteristics with mammals. One such characteristic is its kidneys, of which histology and functions are similar to those of mammals, although unique differences of zebrafish kidneys, such as kidney marrow, should be noted. Moreover, the zebrafish kidney is simpler in structure and easy to observe. For these advantages, zebrafish has been increasingly used as an experimental model for screening nephrotoxicity of chemicals and for understanding related mechanisms. Multiple endpoints of zebrafish model, from functional level, i.e., glomerular filtration, to transcriptional changes of key genes, have been assessed to identify chemical-induced kidney toxicities, and to elucidate underlying mechanisms. The most frequently studied mechanisms of chemical-induced nephrotoxicity in zebrafish include oxidative stress, inflammation, DNA damage, apoptosis, fibrosis, and cell death. To date, several pharmaceuticals, oxidizing agents, natural products, biocides, alcohols, and consumer chemicals have been demonstrated to exert different types of kidney toxicities in zebrafish. The present review shows that zebrafish model can be efficiently employed for quick and reliable assessment of kidney damage potentials of chemicals, and related toxic mechanisms. The toxicological information obtained from this model can be utilized for identification of nephrotoxic chemicals and hence for protection of public health.
Histopathology can reveal toxicant‐induced changes in the structure of a tissue or organ. A prerequisite for histopathological studies is a sound knowledge of the morphology of the anatomical structure in the normal or healthy state. Zebrafish larvae can provide a tool for studies focused on hepatotoxicity at early stages of development; therefore, the fine structure of the organ should be well characterised. To date, liver structure at 72 and 120 hr post‐fertilisation (hpf) has not been reported in detail and this study aimed to fill this scientific gap. A stereological approach allowed for quantitative description of the liver and revealed ultrastructural alterations occurring with time of development. These included a significant increase in the absolute volume of hepatocytes, mitochondria and rough endoplasmic reticulum (rER) during the period of study. The surface area of rER, and of outer and inner mitochondrial membranes also increased. There was no change in the absolute volume of the nuclei. This study provides a quantitative spatial and temporal framework for future research aiming to detect early developmental changes in the liver. Zebrafish larvae can provide a tool for studies focused on hepatotoxicity at early stages of development. In the present study hepatocyte ultrastructure was quantified using a stereological approach at 72 and 120 hr post‐fertilization. This study provides a quantitative spatial and temporal framework for future research aiming to detect early developmental changes in the liver.
Logically, aromatic nitro and amino compounds should be discussed together because their toxic responses are often similar due to a common metabolic intermediate. Synthetically, amines are generally derived from nitro compounds, but in some cases nitro compounds can be prepared through amines when other methods fail to afford specific compounds. There are good and bad attributes to these types of compounds. Some act as sensitizers and contingent on physical properties, may be absorbed through the skin or mucous membranes. They may also cause methemoglobinemia, depending on such factors as the structure and the particular organism. Some members of this class are known as animal and human carcinogens; for humans the urinary bladder is the most prominent target organ. Nevertheless, these compounds and their derivatives have enlivened our world through their use as dyestuff intermediates or as photographic chemicals, they alleviate pain as components of widely used analgesics, and they cushion or insulate us through their use in flexible and rigid foams. Other important uses include production of pesticides, including herbicides and fungicides, as ingredients in adhesives, paints and coatings, antioxidants, explosives, optical brighteners, rubber ingredients, and as intermediates in many other products.
Cytopathological alterations in hepatocytes of fish following exposure to xenobiotic compounds represent a powerful tool to reveal sublethal effects of chemicals and to elucidate underlying modes of action. The present communication reviews the available information about ultrastructural changes in fish liver as well as isolated hepatocytes; whereas the discussion of in vivo effects is primarily focused on data from rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio), the presentation of in vitro data has been restricted to results from experiments with rainbow trout hepatocytes due to a lack of data from studies with hepatocytes from other species. Both in vivo and in vitro exposure to xenobiotics results in sensitive, selective, and, especially in in vitro experiments, extremely rapid responses of hepatocytes, which, however, may be confounded by internal parameters (species, sex, age, hormonal status) and external parameters (temperature, nutrition, duration of exposure). Thus, transfer of results and conclusions from one experiment to another is usually not possible. Likewise, in vitro results may not necessarily be extrapolated to the situation in intact fish, and effects by acute toxic exposure cannot be translated into sublethal effects.
Logically, aromatic nitro and amino compounds should be discussed together because their toxic responses are often similar due to a common metabolic intermediate. Synthetically, amines are generally derived from nitro compounds, but in some cases nitro compounds can be prepared through amines when other methods fail to afford specific compounds. There are good and bad attributes to these types of compounds. Some act as sensitizers and contingent on physical properties may be absorbed through the skin or mucous membranes. They may also cause methemoglobinemia, depending on such factors as the structure and the particular organism. Some members of this class are known as animal and human carcinogens; for humans, the urinary bladder is the most prominent target organ. Nevertheless, these compounds and their derivatives have enlivened our world through their use as dyestuff intermediates or as photographic chemicals, they alleviate pain as components of widely used analgesics, and they cushion or insulate us through their use in flexible and rigid foams. Other important uses include production of pesticides, including herbicides and fungicides, as ingredients in adhesives, paints and coatings, antioxidants, explosives, optical brighteners, rubber ingredients, and as intermediates in many other products.Keywords:Air pollutants;aromatic amino compounds;aromatic nitro compounds;bladder cancer;chloro compounds;databases;inventories
Members of the catadromous eel live in various fresh, brackish and marine habitats. Therefore, these eels can accumulate organic pollutants and are a suitable bioindicator species for determining the levels of organic contaminants within different water bodies. The ecological risk for organochlorine compounds (OCs) in Anguilla japonica with various migration patterns, such as freshwater, estuarine and marine residences, was examined to understand the specific accumulation patterns. The concentrations of HCB, ∑HCHs, ∑CHLs and ∑DDTs in the silver stage (maturing) eel were significantly higher than those in the yellow stage (immature) eel, in accordance with the higher lipid contents in the former versus the latter. The OC accumulations were clearly different among migratory types in the eel. The ecological risk of OCs increased as the freshwater residence period in the eel lengthened. The migratory histories and the lipid contents directly affected the OC accumulation in the catadromous eel species.
Zebrafish have been used predominantly in developmental biology and molecular genetics, but their value in toxicology as well as drug discovery has been recognized. To evaluate the toxicity of a chemical, it is essential to identify the endpoints of toxicity and their dose-response relationships, elucidate the mechanisms of toxicity, and determine the toxicodynamics of the chemical. In addition to detailed toxicological investigations of a single chemical, there also is a need for high-throughput large- scale screening for toxicity of several hundreds of chemicals at a time. In both cases, the zebrafish has numerous attributes. More is probably known about ''what is normal'' in the zebrafish than any other fish species. This includes morpho- logical, biochemical, and physiological information at all stages of early development and in juveniles and adults of both sexes. This makes using the zebrafish ideal for toxicology research where the objective is to identify adverse effects of chemical exposure.
The bioaccumulation of organochlorines (OCs) in the muscle tissue of sea-run (anadromous) and freshwater-resident (fluvial) white-spotted charr (Salvelinus leucomaenis) was determined to assess the ecological risk related to intraspecies variations in diadromous fish life history as they migrate between sea and freshwater. Generally, there were significant correlations between the accumulation of OCs such as DDTs, HCB, HCHs and CHLs. In addition, various biological characteristics, such as total length (TL), body weight (BW) and age, and number of downstream migration (NDM) were correlated. A positive correlation occurred between the lipid content and the OC concentrations. Close linear relationships were found between TL, BW and NDM and the lipid content. Although they are both the same species, the OCs concentrations in the anadromous fish were significantly higher than those in the fluvial individuals. These results suggest that anadromous S. leucomaenis have a higher ecological risk for OCs exposure than the fluvial fish.
Integrierte Sedimentuntersuchungen bieten durch den kombinierten Einsatz von Bioassays, chemischer Analytik und Bestandaufnahmen in situ umfassende Informationen zum Schädigungspotential von Sedimenten, überprüfen zugleich die ökologische Relevanz der Laboruntersuchungen, geben aber keine Informationen über die biologisch wirksamen Substanzklassen. Mittels Bioassay-dirigierten Fraktionierungstechniken können problematische Substanzklassen identifiziert werden. Der direkte Transfer der Ergebnisse auf das Ökosystem ist jedoch problematisch. Daher wird in diesem Projekt versucht, beide Konzepte miteinander zu verbinden, da für eine umfassende Risikoabschätzung sowohl die Überprüfung der ökologischen Relevanz von Laborexperimenten als auch eine Identifizierung der biologisch wirksamen Fremdstoffe notwendig ist. Es werden (1) das Untersuchungskonzept, das Biotests zur akuten Toxizität, dioxinähnlichen, genotoxischen und endokrinen Wirksamkeit beinhaltet, (2) Befunde der integrierten Untersuchungen und (3) Ansätze zu einer Bewertung vorgestellt. Bei einer Flachwasserzone am Neckar erwies sich insbesondere der Fischeitest (mit Danio rerio) mit den Expositionspfaden natives Sediment, Porenwasser und organischer Extrakt als geeignet, das ökotoxikologische Schädigungspotential zu charakterisieren und Hinweise zur Bioverfügbarkeit der partikulär gebundenen Schadstoffe für Fische zu geben. Mittels Bioassay-dirigierten Fraktionierungstechniken (Flüssig/Flüssig-Fraktionierung und säulenchromatographische Auftrennung mit Aluminiumoxid und HPLC) konnten am Beispiel eines mit Klinikabwässern belasteten Fließgewässers weiterführende Informationen zur Identifikation jener Substanzen gewonnen werden, die im Bioassay eine mutagene Wirkung hervorriefen. Die Ergebnisse einer vergleichenden Bewertung werden mithilfe eines einfachen ortsabhängigen Klassifikationssystems für kontaminierte Fließgewässer vorgestellt und einer Bewertung auf der Basis von Fuzzy Logic gegenübergestellt.
Das Ziel der vorliegenden Arbeit bestand darin, durch Einsatz verschiedener biologischer Tests und Endpunkte ein Set von Biotests zu etablieren, welches zur ökotoxikologischen Diagnostik des Belastungszustands kleiner Fließgewässer einsetzbar ist. Zunächst wurde die akute Toxizität von freiem Wasser und wäßrigen Eluaten bzw. acetonischen Extrakten von Sedimenten aus zwei Modellfließgewässern in der Nähe von Stuttgart, dem Krähenbach und der Körsch, die sich in ihrer Belastung durch Schadstoffe deutlich unterscheiden, mit Hilfe der Dauerzellinie RTG-2 aus der Regenbogenforelle ermittelt. Im zweiten Teil dieser Arbeit wurden isolierte Hepatocyten der Regenbogenforelle eingesetzt, um Aussagen zur subakuten cyto- und gentoxischen Wirkung von Wasser- und Sedimentproben zu erhalten. Es folgte schließlich ein Vergleich von biochemischem und cytopathologischem Reaktionsmuster von Primärhepatocyten auf Belastung mit Wasser- und Sedimentproben sowie Schadstoffgemischen, die entsprechend der durchschnittlichen Belastung des stärker kontaminierten Gewässers (Körsch) zusammengesetzt waren. Im dritten Teil wurden Frühstadien des Zebrabärblings eingesetzt, um natives Wasser und Sediment der beiden Fließgewässer in einem Early Life Stage-Test auf ihre akute bzw. subakute Embryotoxizität zu untersuchen. Neben klassischen Parametern wie Mortalität und Schlüpfrate wurden auch cytopathologische Veränderungen in der Leber der Frühstadien berücksichtigt. Die Kombination von Zell- und Embryolarvaltests mit biochemischen, gentoxischen und ultrastrukturellen Untersuchungen erwies sich als geeignet, ein empfindliches Instrumentarium zur ökotoxikologischen Diagnose des Belastungszustands kleiner Fließgewässer bereitzustellen. Insbesondere im Kompartiment Sediment konnte so ein hohes cyto-, gen- und embryotoxisches Potential ermittelt werden.
Ultrastructural alterations in liver and gills of embryonic and larval zebrafish (Danio rerio) following prolonged exposure to waterborne 0.05, 0.5, and 5 mg/L 4-chloroaniline for up to 31 days as well as after a 14-day regeneration period were investigated by means of light and electron microscopy. Acute toxicity was also tested at 25 and 50 mg/L. Survival of zebrafish embryos and larvae was only impaired from 25 mg/L 4-chloroaniline, but-after a transient stimulation following exposure to 0.5 mg/L-4-chloroaniline hatching was retarded after exposure to >/=5 mg/L, and fish displayed increasing rates of abnormal development and pigmentation. In contrast, hepatocytes displayed a time- and dose-dependent response from 0.05 mg/L 4-chloroaniline, including changes in nuclei, mitochondria, peroxisomes, endoplasmic reticulum, Golgi fields, lysosomes, and hepatic glycogen and lipid stores, as well as invasion of macrophages. In gills, dose-dependent effects were evident from 0.5 mg/L 4-chloroaniline and included deformation of secondary lamellae due to vacuolization and desquamation of respiratory epithelial cells in conjunction with dilation of intercellular spaces. Respiratory epithelial cells displayed progressive mitochondrial changes, induction of cytoplasmic myelinated structures, augmentation of lysosomes, and modifications of Golgi fields. Erythrocytes were severely deformed. A 14-day regeneration period was sufficient for almost complete recovery of pathological symptoms in both liver and gills. Only minor volumetric changes in hepatocellular organelles and a limited number of myelinated bodies, lysosomes, and cytoplasmic vacuoles were reminiscent of prior 4-chloroaniline exposure. In both qualitative and quantitative terms, most effects in hepatocytes after exposure of embryonic and larval zebrafish to waterborne 4-chloroaniline are comparable to the reaction of hepatocytes in adult zebrafish liver after prolonged sublethal exposure as well as in larval zebrafish after microinjection. Morphological changes in erythrocytes indicate disturbance of respiration as an additional mode of action of 4-chloroaniline.
In order to evaluate the toxicity of the fungicide triphenyltin acetate (TPTA) on hatching, survival, morphology, and histology of early life stages of zebrafish (Danio rerio), newly fertilized eggs were exposed to concentrations of 0.5, 5, 25, 75, and 100 microg/liter TPTA for 96 h at 28+/-0.5 degrees C. Embryos and larvae were kept under constant observation for up to 7 days and studied with respect to mortality and teratogenic effects as well as histological and cytological alterations in the liver as endpoints of sublethal toxicity of TPTA. Exposure to TPTA caused dose- and time-related effects with respect to all parameters investigated: (1) Hatching was delayed at concentrations >/=0.5 microg/liter TPTA; (2) mortality increased at >/=25 microg/liter TA after 96 h exposure, with TPTA toxicity being higher in larval than in embryonic stages; (3) from >/=25 microg/liter, larvae exhibited skeletal malformation, retarded yolk sac resorption, and edema in the heart and yolk sac regions; and (4) histo- and cytopathological alterations of larval liver included changes in nuclei and mitochondria as well as glycogen depletion from >/=0.5 microg/liter TPTA. The study thus demonstrates not only an elevated sensitivity of zebrafish embryos to TPTA in stages prior to hatching, but also the importance of continuous observation over extended periods after termination of the actual exposure for a comprehensive evaluation of the toxicity of chemical compounds.
Peroxisomes increase in size and number in responsive animals ranging from mammals to marine mussels and fish species when treated with certain compounds named peroxisome proliferators. This phenomenon, known as peroxisome proliferation, is mediated by nuclear receptors termed peroxisome proliferator-activated receptors (PPARs). Three PPAR subtypes have been described (alpha, beta, and gamma) and in mammals PPARalpha is mainly expressed in tissues that catabolize fatty acids, PPARbeta is ubiquitously distributed, and PPARgamma is mainly expressed in the adipose tissue and immune system. The aim of this study was to analyze the tissue distribution of different PPAR subtypes in zebrafish Danio rerio using commercially available antibodies against PPARalpha, PPARbeta, and PPARgamma. In western blots, specific bands were detected at about 58 kDa for PPARalpha and PPARbeta. For PPARgamma the band was detected at 56 kDa. Similar results were obtained in mouse liver homogenates used as positive control, indicating the specificity of the antibodies. Immunohistochemistry was performed in paraformaldehyde-fixed tissue using either microwave or microwave plus trypsin pretreatment for antigen retrieval. In zebrafish, PPARalpha was expressed mainly in liver parenchymal cells, proximal tubules of kidney, enterocytes, and pancreas. PPARbeta showed a widespread distribution and was expressed in the liver, proximal and distal tubules and glomeruli of the kidney, pancreas, enterocytes and smooth muscle of the intestine, skin epithelium, lymphocytes, and male and female gonads. PPARgamma expression was weak in pancreatic cells, intestine, and gonads for both pretreatments. Most of the signal detected was cytoplasmic; only in the cases of PPARalpha and PPARbeta was some nuclear labeling detected in the liver. In mouse tissues, the distribution of PPAR subtypes was similar to that described previously for rats. Our results demonstrate that all three distinct PPAR subtypes are present in zebrafish. The tissue and cellular distribution of PPAR subtypes in zebrafish resembled partly that described before in mammals. Further studies are needed to decipher the functions of PPAR subtypes in zebrafish and other aquatic organisms and particularly their role in regulation of metabolic responses to xenobiotic exposure.
The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1-2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology.
Peroxisome proliferators comprise a heterogeneous group of compounds known for their ability to cause massive proliferation of peroxisomes and liver carcinogenesis in rodents. In recent years it has become evident that other animals may be threatened by peroxisome proliferators, in particular aquatic organisms living in coastal and estuarine areas. These animals are exposed to a variety of pollutants of industrial, agricultural and urban origin which are potential peroxisome proliferators. Both laboratory and field studies have shown that phthalate ester plasticizers, PAHs and oil derivatives, PCBs, certain pesticides, bleached kraft pulp and paper mill effluents, alkylphenols and estrogens provoke peroxisome proliferation in different fish or bivalve mollusc species. The response appears to be mediated by peroxisome-proliferator activated receptors, members of the nuclear receptor family, recently cloned in fish. Based on these results it is proposed that peroxisome proliferation could be used as a biomarker of exposure to a variety of pollutants in environmental pollution assessment. This is illustrated by a case study in which mussels, used worldwide as sentinels of environmental pollution, were transplanted from reference to contaminated areas and vice versa. In mussels native to an area polluted with PAHs and PCBs, peroxisomal acyl-CoA oxidase (AOX) activity and peroxisomal volume density were 2-3 fold and 5-fold higher, respectively, compared to the reference site. When animals were transplanted to the polluted station, with increased concentration of organic xenobiotics, a concomitant significant increase of AOX was recorded. Conversely, in animals transplanted to the cleaner station, AOX activity and peroxisomal volume density decreased significantly. These results indicate that peroxisome proliferation is a rapid (i.e., two days) and reversible response to pollution in mussels. Before peroxisome proliferation can be implemented as a biomarker in biomonitoring programs, a well-defined protocol should be established and validated in intercalibration and quality assurance programmes. Furthermore, the influence of biotic and abiotic factors, some of which are known to affect peroxisome proliferation (season, tide level, interpopulation and interindividual variability), should be taken into consideration. The possible hepatocarcinogenic effects as well as the potential adverse effects on reproduction, development, and growth of peroxisome proliferators are unknown in aquatic organisms, thus providing a challenge for future investigations.
Zebrafish (Danio rerio) has been a prominent model vertebrate in a variety of biological disciplines. Substantial information gathered from developmental and genetic research, together with near-completion of the zebrafish genome project, has placed zebrafish in an attractive position for use as a toxicological model. Although still in its infancy, there is a clear potential for zebrafish to provide valuable new insights into chemical toxicity, drug discovery, and human disease using recent advances in forward and reverse genetic techniques coupled with large-scale, high-throughput screening. Here we present an overview of the rapidly increasing use of zebrafish in toxicology. Advantages of the zebrafish both in identifying endpoints of toxicity and in elucidating mechanisms of toxicity are highlighted.
The goals of the present study were to (1) evaluate the chronic nephropathology caused by a single hexachlorobutadiene (HCBD)-treatment; (2) to characterize the patterns of repair and epithelial regeneration in the goldfish Carassius auratus kidney; and (3) to determine the rate of cell turnover in normal and injured goldfish nephrons
New nephrons developed in goldfish, Carassius auratus, several weeks following hexachlorobutadiene-induced (HCBD) nephrotoxicity. Basophilic clusters of presumptive nephrogenic cells incorporated 5-bromo, 2'deoxyuridine (BrdU) one week after HCBD injection, indicating initiation of DNA synthesis. These clusters, like renal vesicles in the developing kidney, elongated, fused with collecting ducts and developed into immature nephrons during the next 2 weeks. Stereologic quantification showed the volume percent of the kidney occupied by the developing nephrons was greater in HCBD-treated fish 2, 3, 4, and 10 weeks after injection than in the control fish. The presence of large numbers of developing nephrons may provide a marker for renal injury in fish from contaminated waterways.
We examined the metabolism and disposition of aniline, which induces spleen hemangiosarcomas in rats but no tumors in mice, in normal and predosed Fischer 344 rats, and C57BL/6 X C3H F1 mice administered low (50 and 100 mg/kg, respectively) or high (250 and 500 mg/kg, respectively) doses. Of 11 tissues examined, the highest levels of binding of [14C]aniline to DNA were in the kidney, large intestine, and spleen of high-dose rats that had received prior dosing; these tissues had covalent binding indices of 14.2, 4.3, and 3.7 mumol/mol nucleotides/dose, respectively. Protein and RNA were the major macromolecular targets for binding of radioactivity from [14C]aniline. Relative to controls, most tissues from predosed mice (low dose and high dose) showed less binding to protein and RNA; but for most tissues from predosed rats administered 50-mg/kg doses of [14C]aniline, there was more extensive binding. Also relative to controls, binding of radioactivity in the spleen of predosed rats given [14C]aniline (50 mg/kg) was 148% greater for protein and 302% greater for RNA. For rats administered 250 mg of [14C]aniline per kg, however, there were no outstanding differences in binding to RNA and protein between normal and predosed animals. The profiles of urinary metabolites produced by rats and mice were not appreciably different in animals predosed with aniline. For rats, however, the profiles were different for the low and high doses, suggesting that the main metabolic pathway was saturated at the higher dose. p-Acetamidophenyl sulfate represented over 70% of the total radioactivity recovered from the urine of rats dosed with 50 mg of aniline per kg but only 30% in the urine of those dosed with 250 mg/kg. The urine of the high-dose rats contained greater percentages of p-aminophenyl sulfate, p-acetamidophenyl glucuronide, and unconjugated metabolites. In mouse urine, p-acetamidophenyl glucuronide, representing 29 to 32% of the total radioactivity, was the major metabolite. Nevertheless, mice produced more ortho derivatives than did rats, for in acid-treated urine, the ratio of p- to o-aminophenol was 8.1 for rats and 1.6 for mice. Predosing of rats and mice did not change the kinetic values for liver aniline p-hydroxylase or N-hydroxylase but increased the amount of mouse liver cytochrome P-450 from 0.231 to 0.491 nmol/mg protein. For p-hydroxylase of rat liver, the apparent Km value was higher, and the apparent Vmax value lower than in mouse liver. Kinetic values for rat and mouse N-hydroxylase were similar.(ABSTRACT TRUNCATED AT 400 WORDS)
Aniline of unknown purity has been reported to induce spleen hemangiosarcoma in rats. Aniline has been found to be negative in terms of mutagenicity in both bacteria and yeasts. We have found that both commercial (already rather pure) and repurified aniline are clearly positive to a similar extent in inducing DNA damage in vivo in liver and kidney of rats. Both the commercial and repurified product are also clearly positive in induction of sister chromatid exchanges in vivo in male Swiss mice bone marrow cells. Liver, kidney, and bone marrow DNA damage was absent in male Swiss mice.
Hexachlorobutadiene (HCBD), a chlorinated hydrocarbon, is an acute renal toxicant in mammals. Goldfish (Carassius auratus) were given a single i.p. injection of a sublethal dose (500 mg/kg) of HCBD and sampled daily for one week. No damage was observed by light microscopy 6 h post injection. At 24 h, however, cytoplasmic vacuolation and necrosis occurred in the renal tubules. This damage was localized to the epithelium of the second (P2) and third (P3) segments of the proximal tubule. The damage persisted for seven days. By the sixth day the first segment (P1) of the proximal tubule had small cytoplasmic vacuoles. The ratio of kidney to body weight was significantly greater in the treated fish on the fourth day. Gamma glutamyl transpeptidase (GGT), a histochemical marker of proximal tubule brush border in mammals, was demonstrated in the goldfish kidney. Intense staining was noted only in P2 and P3. GGT staining was also present in the lumen of the damaged, vacuolated tubules of HCBD-treated fish.
In order to evaluate the substance-specificity of ultrastructural criteria in isolated fish hepatocytes as endpoints in toxicological studies, liver cells isolated from rainbow trout (Oncorhynchus mykiss) by collagenase perfusion were exposed in vitro to sublethal concentrations of dinitro-o-cresol (DNOC: 0, 0.7, 7.2, 72.2 μmol/1) and 2,4-dichlorophenol (2,4-DCP: 0, 6.1, 61.3, 613 nmol/1) for 1–5 days. With either toxicant, earliest cytological modifications could be detected after 1 day at the lowest test concentrations. Ultrastructural analysis documents a complex, substance-specific combination of dose- and time-dependent effects. Evaluation of single cytological alterations induced by DNOC and 2,4-DCP reveals that each cytopathological reaction consists of both unspecific and substance-specific changes. Unspecific symptoms comprise disturbance of intracellular compartmentation, condensation, marginalization and redistribution of heterochromatin, reduction and reorganization of RER cisternae, proliferation of lysosomes, glycogenosomes, myelinated bodies and large electron-lucent vacuoles with heterogeneous contents, as well as glycogen depletion. Transformation of Golgi cisternae into concentric membrane whorls is specific to intoxication by DNOC, whereas the reaction to 2,4-DCP is characterized by unusual heterochromatin distribution patterns and formation of mitochondrion aggregations. Due to their general character, unspecific changes are recommended as rapid biomarkers of contamination by chemicals, whereas specific changes might be useful for the differential diagnosis of the toxicants.
The morphological alterations of hepatocytes of female zebrafish,Brachydanio rerio, and fingerling rainbow trout,Salmo gairdneri, following prolonged exposure to 0.04, 0.2 and 1 mg/L of 4-chloroaniline were investigated by means of light and electron microscopy. Changes in peroxisomes were visualized by cytochemical demonstration of catalase activity after incubation in the alkaline diaminobenzidine medium. The amount of storage products was illustrated by the silver impregnation technique.
In a dose-dependent manner, the reaction of female zebrafish liver is characterized by a disturbance of hepatocytic compartmentation, progressive fenestration and fractionation of the rough endoplasmic reticulum (RER), a decrease in the number of peroxisomes and catalase activity, stratified inclusions in mitochondria, and an augmentation of lysosomes and myelinated bodies. Trout hepatocytes display nuclear inclusions, fractionation and vesiculation of the RER, and an increase in mitochondria, but a decrease of peroxisomes and catalase activity. Whereas glycogen stores are exhausted at 1 mg/L 4-chloroaniline, lipid deposits are amplified. An elevated rate of hepatocytic mitosis as well as the occurrence of glycogen-condensing cells probably derived from hepatocytes indicate the induction of proliferative processes in trout liver.
Evaluation and comparison of results with earlier reports suggest that despite the unspecificity of some alterations the combination of pathological symptoms yields a syndrome specific of the species and the substance studied. As a consequence, histological and cytological investigations are recommended as a routine supplement in an integrated test schedule for the assessment of sublethal effects of pollutants in the aquatic environment.
Morphological alterations of the liver of zebra fish (Brachydanio rerio) following prolonged exposure to 0.1, 1 and 5 mg · l−1 4-nitrophenol (4-NP) were investigated by means of light and electron microscopy. Based on marked sexual dimorphism in control animals, liver recactions were both sex- and dose-dependent. Whereas at 0.1 mg·1−1 only minor changes could be revealed, there were numerous structural modifications at 1 mg·1−1: Whereas male fish primarily react with a proliferation of smooth endoplasmic reticulum, female fish display a high degree of fenetrastion within cisternate of the rough endoplasmic reticulum. Both sexes exhibit infiltration of macrophages and lymphocytes, an increase in the number of binucleate cells, of lysosomes, autophagosomes and myelinated bodies, but a depletion of hepatic glycogen. At 5 mg·1−1, deformations of the nuclear membrane and partial lysis of mitochondria could be observed. At 1 and 5 mg·1−1 4-NP about 25% of the animals investigated showed symptoms of degenerative transformations of the liver tissue into huge multinucleate cell masses with completely different ultrastructure.The evaluation of the results leads us to the conclusion that for toxicological studies on the effects of ‘priority pollutants’ histological and cytological investigations should be incorporated into an integrated methodological scheme.
In a full life cycle test, newly hatched eggs of zebra fish, Brachydanio rerio, were reared to sexual maturity under continuous exposure to 40, 80, 110, 130, and 150 μg/liter lindane, γ-hexachlorocyclohexane. The effects of lindane were investigated by recording behavior and survival of the F0- and F1-generation as well as morphological alterations in liver ultrastructure of F0. Changes in peroxisomes were visualized by cytochemical staining for catalase activity with diaminobenzidine. Whereas behavioral changes can already be observed at 40 μg/liter, survival and number of eggs in F0 as well as survival and growth of F1 are unaffected by up to 80 μg/liter lindane. At concentrations ⩾ 110 μg/liter, survival of larvae is reduced already after 35 days, and mortality is 100% after 3 months. From 40 μg/liter, liver ultrastructure displays a microvesicular fatty vacuolation (steatosis) characterized by lipid deposition within the cisternae of the RER. At 40 μg/liter, this highly specific pathological change is accompanied by accumulation of hepatic macrovesicular triglyceride droplets, glycogen depletion, and the occurrence of club-shaped mitochondria. Additional alterations at 80 μg/liter comprise proliferation of SER in males and progressive fractionation of RER in females, stacking of club-shaped mitochondria, a conspicuous decrease in peroxisomal catalase activity, infiltration of macrophages into the liver parenchyma, and a significant stimulation of hepatocytic mitosis. Among several substances tested so far in zebra fish (4-nitrophenol, 4-chloroaniline 3,4-dichloroaniline, atrazine, lindane), lindane is the only compound inducing behavioral changes and hepatic steatosis in conjunction with a reduction in fertility. With regard to the relative sensitivity of the methods applied, behavioral and cytological studies appear more responsive to lindane exposure than survival studies.
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.
In order to evaluate the suitability of ultrastructural criteria in isolated fish hepatocytes as endopoints in toxicological studies, liver cells isolated from rainbow trout (Oncorhynchus mykiss) by collagenase perfusion were exposed in vitro to sublethal concentrations (1, 3, 10 mg/l) of the reference toxicant 4-chloroaniline (4-CA) for up to 8 days. Cytopathological alterations in isolated cells were recorded after 1, 3 and 5 days and compared to hepatopathological effects induced by prolonged in vivo exposure of rainbow trout. Whereas trypan blue exclusion failed to reveal any effect of 4-CA exposure, cytological modifications could be detected as early as 1 day at 1 mg/l. Ultrastructural analysis unequivocally documented a complex set of sublethal effects in a dose- and time-dependent manner, including changes in all hepatocytic organelles. Evaluation and comparison of in vitro results with hepatocellular effects in intact rainbow trout suggested major correspondence in some fundamental hepatocellular functions such as intracellular compartmentation, number of mitochondria and lysosomes, ultrastructural organization of the RER, as well as the secretory activity of the Golgi fields. Other symptoms, e.g., ER transformation into myelinated structures, most likely indicated toxicant-induced premature ageing of isolated hepatocytes. Apparent discrepancies between hepatocytic reactions in vivo and in vitro must be attributed to differential activation of metabolic pathways or the modulation of basic hepatocellular responses by external systemic factors.
To characterize the risk that polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs) pose to salmonid early life stage survival, we developed a method to expose rainbow trout (Oncorhynchus mykiss) eggs to graded doses of PCDD, PCDF, and PCB congeners, using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a prototype. Rainbow trout eggs were injected 24–50 h post-fertil ization with 0.2 μl of 50 mM phosphatidylcholine (PC) liposomes (control) or 0.2 μl of 5–7 graded doses of TCDD incorporated into 50 mM PC liposomes. Injection volume never exceeded 0.6% egg volume. Immediately following injection, the injection site was sealed with Super glueR, resulting in 92–97% of TCDD dose retained by the egg. Following both egg injection and waterborne egg exposure. TCDD toxicity in rainbow trout was manifested by half-hatching mortality but predominantly by sac fry mortality associated with hemorrhages, pericardial edema, and yolk sac edema. TCDD LD50s, following injection and waterborne exposure of rainbow trout eggs, were 421 (331–489) and 439 (346–519) pg TCDD/g egg (LD50, 95% fiducial limits), respectively. As in rainbow trout, TCDD toxicity in lake trout (Salvelinus namaycush) following the same two routes of exposure was manifested by half-hatching mortality but predominantly by sac fry mortality preceded by hemorrhages and yolk sac edema. LD50s, based on the dose of TCDD in lake trout eggs, were 47 (21–65) and 65 (60–71) pg/g following injection and waterborne exposure, respectively. The egg injection method is ideal for assessing the relationship between early life stage mortality in rainbow trout and graded egg doses of individual PCDD, PCDF, or PCB congeners.
Cultured fish cells can be used in a variety of cytotoxicity and genotoxicity assays for the preliminary testing of environmental chemical hazards that may be hazardous to the aquatic biota. Such assays can also be used to evaluate synergistic and antagonistic interactions between combinations of test agents and to establish structure-activity relationships for series of related chemicals. A range of fish cell lines are available for use in such assays and a variety of endpoints may be used. To detect toxicants that require bioactivation the chosen cell line must have significant P-450 activity, or a metabolizing component must be incorporated into the assay. Fish cells in culture respond to the same chemical mutagens and clastogens that are genotoxic to mammalian cells in culture. However, since fish cells in culture are eurythermic, they represent a unique system for studying temperature as a parameter in mediating the genotoxicity and the cytotoxicity of a test agent.
Aquatic toxicology, ISSN 0166-445X, vol. 26, 307-316 In this experimental study a microinjection technique for the administration of lipid-soluble xenobiotics in rainbow trout embryos was applied. One week prior to hatching, rainbow trout embryos were injected in the yolk-sac with commercial blends of polychlorinated biphenyls (PCBs), polychlorinated naphthalenes (PCNs), polychlorinated paraffins (PCPs) and polybrominated diphenyl ethers (PBDEs). Six weeks later, the morphology of the liver was examined, and the hepatic cytochrome P450-dependent ethoxyresorufin-O-deethylase (EROD) activity was determined in the swim-up fry. A dose-dependent induction of the EROD activity was found for the PCBs and the PCNs, indicating that application of the microinjection technique in fish embryo bioassays, combined with studies of liver morphology and biochemistry, facilitate detection of biological responses to certain lipid-soluble substances.
The response of the channel catfish liver to subacute exposure of polychlorinated biphenyls was evaluated using electron microscopic and biochemical techniques. After 21 days, treated fish displayed elevated microsomal enzyme activities. Morphologically, the liver produced several patterns of alteration involving the endoplasmic reticulum (ER). Structural alterations included an increase in tubular smooth ER, production of parallel stacks of smooth ER showing continuity with rough ER, and membranous whorls. Biochemical and morphologic findings were correlated in exposed livers, and the relationship of these findings to similar studies in other species of fish is discussed.
Rainbow trout (Salmo gairdneri) were fed dietary levels of 0-, 1-, 10-, and 100-ppm Aroclor 1254 for 229 to 330 days. The ultrastructural features of control livers were similar to those of other animals, but rainbow trout liver differed from mammalian liver in an apparent absence of Kupffer cells. Also small dense duct-type cells were present that formed a transition zone, combining with hepatocytes to form canaliculi; alone they formed preductules and in combination with cells of lower density they formed ductules. Microtubules were found in the vicinity of canaliculi and less frequently adjacent to the plasma membrane of other cell surfaces. They were not seen at the sinusoidal border as they are in mammalian liver. Peroxisomes lacked nucleoids. There were no ultrastructural liver changes in the 1-ppm group. The most frequently encountered alterations in both the 10- and 100-ppm groups were those involving the nucleus. These included irregular and bizarre nuclear outlines, separation of nucleolar components, and large nuclear pseudoinclusions. Other frequently encountered changes were slight increases in smooth endoplasmic reticulum, altered rough endoplasmic reticulum, increased lysosomes, reduced and altered glycogen, increased lipid, and hypoxic vacuoles. Seen also were concentric membrane arrays, myelin figures in intercellular spaces and Golgi cisternae, and bundles of fine tubules throughout the cytoplasm.
In order to elucidate cytopathological alterations in hepatic and intestinal cells, immature rainbow trout (Oncorhynchus mykiss) were exposed for five weeks to 10, 20, 40, and 160 micrograms/l of the herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine; model 1). For comparison, ultrastructural changes in female zebra fish (Brachydanio rerio) liver were studied after exposure to 100, 1,000 and 10,000 micrograms/l atrazine for three months (exposure from egg stage to sexual maturation; model 2). Neither epithelial nor glandular cells in the gastrointestinal tract of rainbow trout reveal cytological modifications following exposure to atrazine. In contrast, hepatocytes of rainbow trout and zebra fish clearly display dose-dependent and species-specific cytopathological effects at 40 and 1000 micrograms/l, respectively. In rainbow trout (model 1), rough endoplasmic reticulum (RER) appears of particular diagnostic value for the effects of atrazine, since it already shows a full spectrum of cytological alterations after 40 micrograms/l, and since in cells without RER modifications no further cytopathological symptoms can be revealed. At 40 micrograms/l atrazine, further changes include disturbance of the intracellular compartmentation, increased heterogeneity of mitochondria (longitudinally arranged cristae, branching, size), formation of myelinated bodies as well as immigration of macrophages and granulocytes along the biliary system and the space of Disse. The separation of peripheral storage areas from the central organelle-containing cytoplasm is no longer evident at 80 micrograms/l, and the phagocytic activity of invading macrophages is drastically increased. Following exposure to 160 micrograms/l atrazine, additional pathological changes comprise clubshaped deformation of mitochondria, formation of myelinated bodies in the intermembranous space of mitochondria, increase of degranulated ER cisternae and lysosomes, as well as perisinusoidal accumulation of lipid droplets. Deformation of the nuclear envelope, elevated mitotic activity and an increased number of nuclei with two or more nucleoli indicate interactions between atrazine and the nucleus. In the liver of female zebra fish (model 2), atrazine-induced alterations are limited to increased parenchymal variability, disturbance of the intracellular compartmentation, partial RER fractionation and vesiculation, club-shaped deformation of mitochondria and an increase in the number of lysosomes, myelinated bodies and invading macrophages at 1000 micrograms/l atrazine. After three months at 10,000 micrograms/l, mortality of zebra fish is increased to 100%. According to cytopathological alterations of hepatocytes following long-term exposure, susceptibility of the test model rainbow trout to atrazine appears higher than that of the model zebra fish.(ABSTRACT TRUNCATED AT 400 WORDS)
Liver cytotoxic alterations of adult medaka (Oryzias latipes) following short-term bath exposure (48 hr) to 500 mg/L diethylnitrosamine (DEN) were studied (days 3-21) by electron microscopy and cytochemistry. Control medaka displayed hepatic sexual dimorphism as described for other sexually active fish. Following DEN exposure, decreased glycogen stores with loss of cellular compartmentation obscured sexual dimorphism. A spectrum of organelle alterations, previously not reported in livers of fish, was seen. Early changes in hepatocytes included: nuclear lipid inclusions, nucleolar changes, decreased amounts of granular endoplasmic reticulum (GER), increased fractionation and steatosis of GER, proliferation of smooth ER and lysosomes, reduction in number and content of particulate lipoproteins and vitellogenin in Golgi vesicles, and reduction in number and staining intensity of peroxisomes. At day 14 and/or 21, partial to complete reversal of the above alterations indicated hepatic recovery, and fewer necrotic cells were seen at day 21 versus day 14. Lesions that did not resolve during this study were altered mitochondria and areas of spongiosis hepatis that developed at day 8 and continued to increase throughout the study. Infiltration of lymphocytes, granulocytes, and large numbers of macrophages were late changes. The description, timing, and duration of lesions are of value for consideration as biomarkers of exposure and effect in aquatic toxicology.
Toxicity and histopathological effects of tributyltin chloride (TBT) were studied in early life stages of minnows Phoxinus phoxinus. Eggs and yolk sac fry (newly hatched larvae) were exposed in a static-renewal procedure to aqueous TBT concentrations ranging from 0.82 to 19.51 micrograms/L for 3 to 10 days at 16 degrees C and 21 degrees C, respectively. Aqueous TBT concentrations were determined by capillary GC-FPD and revealed a concentration decrease during the static phase. TBT exposure led to mortality, behavioral, gross morphological and histopathological effects. In larvae, increased mortality, deformation of body axis, paralysis and opaque eyes occurred at 4.26 micrograms/L TBT and higher both in the embryonic-larval and larval exposure. Histological changes were evident at initial TBT concentrations of 0.82 up to 19.51 micrograms/L, and were more pronounced after embryonic-larval exposure than after larval exposure. Degenerative alterations occurred in skin, skeletal muscle, kidney, corneal epithelium, lens, pigment layer of the retina and choroid, retina, and CNS including spinal cord. Hydropic vacuolation of the cytoplasm and, in more pronounced cases, irreversible nuclear alterations such as pycnosis, karyorrhexis and karyolysis were also evident. Exposure to 0.82 micrograms/L TBT resulted in alterations in skin, muscle and kidney, with greater effects occurring at 21 degrees C than at 16 degrees C. Toxicity was significantly reduced in the presence of sediment. The observed histopathological effects suggest that early life stages of fish may be negatively affected in environments that are considerably polluted by TBT.
Fertilized eggs of zebrafish were exposed under flow-through conditions to several concentrations of the following pesticides: lindane 40, 80, 110, 130, and 150 micrograms/liter; atrazine 300, 1300, and 9100 micrograms/liter; deltamethrin 0.5, 0.8, and 1.2 micrograms/liter. Hatching, abnormalities in development (external deformations, edema, etc.), and mortality were recorded over a period of 35 days. At the end of the experiment, the body lengths of the fish were measured. Survival of juvenile fish after 35 days was reduced by increasing concentrations of all xenobiotics tested: lindane enhanced the mortality from 110 micrograms/liter and atrazine from 1300 micrograms/liter, and deltamethrin showed an effect even at the lowest test concentration (0.5 micrograms/liter). Other parameters were affected differently: hatching rate was reduced only by deltamethrin (from 0.8 micrograms/liter): lindane caused a decrease in growth (40 micrograms/liter) but had no effect on the other parameters. Atrazine increased the number of deformations and edema (1300 micrograms/liter) but did not influence hatching rate and growth. The sensitivity of the early life stages to the pesticides was compared with acute toxicity data (LC50) of adult zebrafish. The early life stages were less sensitive to lindane (118 versus 75 micrograms/liter), whereas in the case of atrazine (1300 versus 37,000 micrograms/liter) and deltamethrin (0.5 versus 2 micrograms/liter; 0.5 micrograms/liter was the lowest concentration tested) larvae were more sensitive.
The toxicity of mercury (HgCl2), copper (CuCl2: 5 H20), nickel (NiSO4: 6 H2O), lead (Pb(CH3COO)2: 3 H2O) and cobalt (CoCl2: 6 H2O) was studied under standardized conditions in embryos and larvae of the zebrafish, Brachydanio rerio. Exposures were started at the blastula stage (2-4 h after spawning) and the effects on hatching and survival were monitored daily for 16 days. Copper and nickel were more specific inhibitors of hatching than cobalt, lead, and mercury. Nominal "no effect" concentrations determined from the dose-response relationships (ZEPs, Zero Equivalent Points) for effect on hatching time were 0.05 microgram Cu/L, 10 micrograms Hg/L, 20 micrograms Pb/L, 40 micrograms Ni/L and 3,840 micrograms Co/L, and those for effect on survival time were 0.25 microgram Cu/L, 1.2 micrograms Hg/L, 30 micrograms Pb/L, 80 micrograms Ni/L, and 60 micrograms Co/L. The "no effect" concentrations for Ni, Hg and Pb are consistent with previously reported MATC values for sensitive species of fish. The "no effect" concentrations for copper are 1-2 orders of magnitude lower than previously reported values. The major reason for the latter discrepancy was considered to be the absence of organics that can complex copper ions in the reconstituted water that we used, which had a hardness of 100 mg/L (as CaCO3) and a pH of 7.5-7.7. Unexposed controls were started with embryos from different parental zebrafishes and the parental-caused variability in early embryo mortality, median hatching time and median survival time were estimated.
An early life-stage (ELS) test was conducted with the zebrafish and 3,4-dichloroaniline (3,4-DCA) in eight laboratories. Based on the results from all eight laboratories, an LOEC of 200 micrograms 3,4-dichloroaniline/liter applies for the early life stages of the zebrafish. Effects observed were reduction of the survival rate and malformations. If the 100 micrograms/liter concentration additionally tested by one laboratory is included in the assessment, an LOEC of 100 micrograms 3,4-DCA/liter is obtained for the survival rate and increase in length. The NOEC is 20 micrograms/liter. The present results of a comparative laboratory study with the zebrafish show that a 42-day ELS test can be conducted with this species of fish, and affords meaningful results.
Structural and functional alterations in hepatocytes of the European eel, Anguilla anguilla, following a 4-week-exposure to 5, 50, and 250 micrograms/liter dinitro-o-cresol (DNOC) were investigated by means of electron microscopy and biochemistry and compared to liver pathology in eels exposed to the chemical spill into the Rhine river at Basle in November 1986. Whereas phenological parameters (growth, condition factor) are unaffected, ultrastructural and biochemical alterations are detectable at greater than or equal to 50 and 5 micrograms/liter DNOC, respectively. Structural modifications include: rounding-up of the nuclei; fractionation and reduction of the rough endoplasmic reticulum; proliferation of the smooth endoplasmic reticulum (SER), mitochondria, peroxisomes, and lysosomes; bundles of rod-shaped SER profiles; annulate lamellae; membrane whorls within mitochondria; crystallization of the peroxisomal matrix and glycogen bodies; glycogen depletion and lipid augmentation. Structural changes can be correlated to an increase in hepatic lipid and protein contents as well as stimulation of mitochondrial (cytochrome c oxidase), peroxisomal (catalase, allantoinase, uricase), lysosomal (arylsulfatase), and microsomal (esterase) enzymes. An increase in NADPH-cytochrome c reductase and cytochrome P450 as well as UDP-glucuronyltransferase and arylsulfotransferase activities in the microsomal fraction document an induction of hepatic biotransformation as a functional correlate to SER proliferation. Maximum inducibility of biotransformation enzymes at 50 micrograms/liter indicates a biphasic, concentration-dependent reaction of eel liver. Comparison of DNOC-induced effects with liver pathology in eel exposed to the chemical spill in 1986 reveals striking similarities so that DNOC may not be excluded as a possible factor in the fish kill in the Rhine river.
The effects of the herbicide atrazine (2-Chlor-4-ethyl-amino-6-isopropyl-amino-s-triazine) on the kidney of rainbow trout (Oncorhynchus mykiss) were studied by exposing them to sublethal concentrations of 1.4 and 2.8 mg atrazine per liter of water for 96 h (acute exposure) respectively to 5, 10, 20, 40, and 80 μg/L for a period of 28 days (chronic exposure).
Alterations of the different components of renal corpuscles and of renal tubules, as well as an increase in cells with mitotic figures in renal hemopoietic interstitium were constant features at lower chronic (5, 10, 20, 40 μg/L) exposure; necrosis of endothelial cells and renal hemopoietic tissue were prominent at concentrations of 80 μg/L, and 1.4 and 2.8 mg/L atrazine.
This study describes a long-term test over three generations, using zebrafish (Brachydanio rerio) as the test species and concentrations of 1, 0.2, and 0.04 mg/L 4-chloroaniline (CA) as a model substance. The effect of the compound on the ecologically important parameters reproduction and growth was the focus of interest. Reduction in egg release by fish raised under CA was the most sensitive parameter in the test. Compared to the toxic threshold concentration for growth (0.4 mg/L), egg release was affected by a ten-fold lower concentration (0.04 mg/L). This study demonstrates that a long-term test is still the most appropriate method to assess the chronic toxicity of a substance on fish. A chronic toxicity test is proposed which comprises two generations, with the zebrafish as test species.
A modular apparatus and technique for the injection of salmonid fish embryos with chemical carcinogens are described. A key feature of the methodology is the relative ease of routine through-the-eggshell injection, into the yolk sac of living salmonid fish embryos, inside the "eyed-stage" egg. The procedure is sufficiently rapid that 2 persons working as a team can give injections to 200 embryos per hour. The injection per se induces low mortality, i.e., optimal net survival rates (controls given an injection of dimethyl sulfoxide vs. those not given an injection) in the range of 70-90%. Because only small amounts of chemical are handled in relatively dilute form, the exposure method poses low risks to both the experimentalist and the environment. Preliminary results in a test of 4 carcinogens that differed widely in their structures and requirements for metabolic activation indicated that hepatocellular neoplasms were induced in rainbow trout (Salmo gairdneri) in response to 100 ng aflatoxin B1 (CAS: 1162-65-8)/egg, 1 microgram N-methyl-N'-nitro-N-nitrosoguanidine (CAS: 70-25-7)/egg, and 10 micrograms benzo[a]pyrene (CAS: 50-32-8)/egg. Nine months after exposure, liver neoplasms were observed in 25, 21, and 9%, respectively, of the rainbow trout, but no neoplasms were observed in rainbow trout exposed to 100 micrograms dimethylnitrosamine (CAS: 62-75-9)/egg. Liver neoplasms were also induced in 17% of coho salmon (Oncorhynchus kisutch) given an injection as embryos of 90 ng aflatoxin B1/egg or 5 micrograms N-methyl-N'-nitro-N-nitrosoguanidine/egg.
Extracts prepared from oil refinery effluents (soxhlet and XAD-2) were tested for carcinogenic potential by means of the rainbow trout (Salmo gairdneri) embryo-injection bioassay. No neoplasms were detected in fish given injections of refinery extracts alone (with and without exogenous rat S-9 activation). Refinery extracts coinjected with aflatoxin B1 induced elevated frequencies of hepatic neoplasms. This cocarcinogenic effect was most pronounced when aflatoxin B1 was preincubated with rat S-9 prior to injection. Effluent extracts coinjected with a direct-acting carcinogen [N-methyl-N'-nitro-N-nitrosoguanidine (CAS: 56-57-5)] did not increase the incidence of hepatic neoplasms (with and without exogenous S-9 activation).
Aniline (A) and its monochlorophenyl derivatives (2-CA, 3-CA and 4-CA) are widely-used chemical intermediates. In the present study, the in vivo and in vitro nephrotoxic potential of these compounds was assessed in Fischer 344 rats. In the in vivo experiments, rats were administered a single intraperitoneal (i.p.) injection of an aniline (0.4, 1.0 or 1.5 mmol/kg) or 0.9% saline (2.0 ml/kg, i.p.), and renal function monitored at 24 and 48 h. 2-CA was the only compound tested which decreased urine volume, elevated the blood urea nitrogen (BUN) concentration and depressed both basal and lactatestimulated p-aminohippurate (PAH) accumulation by renal cortical slices at the 1.0 mmol/ kg dose. Similar results were produced following 3- and 4-CA administration, but these compounds required a dose of 1.5 mmol/kg. Aniline had little effect on renal function at the doses used in this study. In the in vitro experiments, 2-CA (10(-4) M or greater) depressed basal PAH accumulation. Tetraethylammonium (TEA) uptake was decreased by all compounds with an incubate concentration of the aniline at 10(-3) M. Lactatestimulated PAH uptake was not decreased by any test compound. These results indicate that chlorine substitution on the phenyl ring of aniline enhances nephrotoxic potential, and that 2-substitution produces the greatest increase.
Effect of a herbicide, paraquat (1,1'-dimethyl-4,4'-bipyridilium-dichloride), the fungicide copper sulphate, and zinc chloride was studied on the histological structure of liver, kidney and gill of three fish species with different feeding habits, viz.: a herbivorous, silver carp (Hypophthalmichthys molitrix); an omnivorous, common carp (Cyprinus carpio L.) and a carnivorous, sheatfish (Silurus glanis L.). The organs were studied electron microscopically after fixation according to Karnovsky. The toxic effect manifested itself characteristically on the respective species, regardless of the type of the chemical applied and the species specificity. Upon the effect of the treatments applied the cytoplasm of the respiratory cells of the gill became electron transparent and the cytoplasmic organelles disappeared almost totally. In the chloride cells showing focal necrosis, residuals of nuclear, mitochondrial and endoplasmic origin were seen. Pillar cells and the pericytes remained intact. In the nucleus of the liver cells, electron dense heterochromatin was not present. The degree of the damage in the liver cells was indicated by swollen mitochondria with electron transparent matrix and by dilatation and vacuolation of the endoplasmic reticulum system. Epithelial cells decreased in electron density, the endoplasmic reticulum was vesiculated, mitochondria were swollen. Leucocytes increased in number, and empty vacuoles and vacuoles filled with dense granules appeared in them during toxicosis. Copper sulphate or paraquat increased serum transaminase enzyme activities (glutamic acid-oxalacetic acid transaminase, glutamic acid-pyruvic acid transaminase) in all the three fish species. These damages can cause serious disturbances in energy uptake and secretion processes of fish.
Liver tumors were induced in rainbow trout (Salmo gairdneri) 1 year after carcinogens were microinjected into embryos. Neoplasms were induced by a single injection of 13 and 25 ng per egg of aflatoxin B1 (CAS: 1162-65-8), 500 ng per egg of 7,12-dimethylbenz[a]anthracene (CAS: 57-97-6), and 250 ng per egg of 2-anthramine (CAS: 613-13-8). Over 70% of [3H]benzo[a]-pyrene (CAS: 50-32-8) injected into eggs was retained in hatched embryos, 120 hours postinjection. Exogenous activation of test compounds with the use of rat liver microsome preparation (S-9) increased the incidence of liver tumors in fish given injections of 25 ng aflatoxin B1. The amount of chemical required for the embryo injection assay was comparable to that required for the Ames bacterial mutagenesis assay.
In 1960 and 1961, linuron and monolinuron were the first substituted 3-aryl-l-methoxy-l-methyl-ureas that were introduced as herbicides in agriculture. Because of their favorable physical, chemical, and toxicological properties, particularly on account of the selectivity of their phyto-toxic effects, they have found world-wide application in many fields of plant production. Their persistence in soil, plants, and the environment is limited to such a degree that, indeed, the desired herbicidal effects are not affected, yet the formation of undesirable residues in food or feed, as well as any accumulation in animate and inanimate nature, are avoided.
21 compounds from different chemical classes were quantitatively compared for their carcinogenic potency according to 4 parameters: (1) potency in inducing covalent binding with DNA in vivo; (2) potency in inducing alkaline DNA fragmentation after treatment in vivo; (3) acute toxicity; (4) mutagenic potency in the Ames test. Establishing well-defined conditions for normalization of the different types of data and determination of the set that had to be submitted to statistical analysis appeared to be a difficult task, for which only compromise solutions were possible. A statistical analysis of the data suggested that all parameters considered were correlated with carcinogenic potency. However, we found that there are about 3 chances to 1 that carcinogenicity is better correlated with DNA covalent binding in vivo than it is to mutagenicity in the Ames test. With due precautions, even acute toxicity could be of predictive value. DNA adducts and DNA fragmentation, both in vivo, appeared to be 2 parameters strongly correlated between them. From a multivariate statistical analysis it appeared that: (1) a significant improvement of quantitative predictability is in principle obtainable with a battery of short-term test; and (2) the improvement is obtainable only if the short-term tests considered, while all correlated with carcinogenicity, are relatively independent amongst themselves.
Mouse liver microsomal oxidases convert diuron [ArNHC(O)N(CH3)2; Ar = 3,4-dichlorophenyl] to seven metabolites modified only at the dimethylamino moiety [-N(CH3)CH2OH,-N(CH3)CHO,-N(CH2OH)2,-NHCH3,-NHCH2OH,-NHCHO, and -NH2] and to an N-hydroxy derivative [ArN(OH)C(O)NHCH3]. Linuron [ArNHC(O)N(OCH3)CH3] in this system yields the corresponding -N(OCH3)CH2OH,-NHCH3, and -NHOCH3 derivatives. Microsomal metabolism of methazole requires NADPH and gives primarily desmethylmethazole under aerobic conditions and ArNHC(O)NHCH3 under nitrogen. Most of these metabolites are also detected in spinach leaves treated with [14C]diuron and [14C]methazole. Some 3-hydroxy-1-methylurea from methazole metabolism in spinach is reduced to the 1-methylurea while the remainder is conjugated as the N-O-β-D-glucoside and its 6-O-malonyl ester (identified by 1H and 13C NMR). Synthesis procedures, spectroscopic data, and potencies as Hill reaction inhibitors are given for many of the metabolites and derivatives which are useful in their identification. Methazole appears to be a proherbicide while diuron and linuron act directly and via metabolites as photosynthetic inhibitors.
Investigations of acute and subacute atrazine toxicity in carp (Cyprinus carpio L.) were carried out. Acute toxicity was investigated in a semi-static test during a 96-hr exposition. The estimated LC-50 value was 18.8 mg/l. Subacute toxicity was investigated by exposing fish (carp) to different atrazine concentrations (1.5, 3.0, and 6.0 mg/l) for 14 days. Biochemical and histopathological changes in certain organs and tissues were investigated. The results show that atrazine leads to changes of varying intensity depending on the parameter tested, the organs and tissues examined, as well as the atrazine concentration. Biochemical changes were most prominent in the alkaline phosphatase, glutamic-oxaloacetic transaminase, and glutamic-pyruvic transaminase activities whereas the most severe histopathological changes were observed in the gills.
Using a static-renewal procedure, effects of triphenyltin chloride (TPT) on hatching, survival, and morphology were assessed in early life stages of European minnows Phoxinus phoxinus. Embryonic-larval exposure at 16 and 21 degrees C, and larval exposure at 16 degrees C were compared. In the embryonic-larval exposure at 16 degrees C, hatching was delayed and hatching success decreased at 15.9 micrograms/L. Mortality increased at > or = 3.9 micrograms/L TPT, and complete mortality occurred after 7 and 9 days at 15.9 and 5.1 micrograms/L, respectively. Mortality was higher at 21 degrees C that at 16 degrees C. Triphenyltin was more toxic to fish in larval stages. The induced effects were dose related, mortality increased at 1.8 microgram/L after 3 days, and was total after 5 days at 10.6 micrograms/L. In all high TPT exposures, larvae developed skeletal malformations (bent tails), showed impaired swimming behavior or paralysis, and eyes became opaque. Marked histopathological alterations were found. Degenerative hydropic vacuolation of the cytoplasm were evident in skeletal muscles, skin, kidneys, corneal epithelium, lens, pigment layer of the retina and choroid, retina, and CNS including spinal cord. In severe cases, nuclear changes including pycnosis and karyorrhexis occurred. The observed toxicity of TPT was similar to that of tributyltin, but TPT acted more selectively on the lens and CNS, whereas other tissues were less affected. The study indicates that Phoxinus phoxinus larvae are negatively affected at peak TPT concentrations found in polluted environments.
Cytopathological alterations in proximal (PS I, PS II) and distal segments (DS) of rainbow trout (Oncorhynchus mykiss) renal tubules following exposure to 0, 10, 20, 40, 80, and 160 micrograms/liter atrazine for 4 weeks were investigated by means of electron microscopy. Cellular responses were clearly dose-dependent with a gradual increase in variability and intensity of effects. Ultrastructural modifications in PS I and II were observed from 10 micrograms/liter atrazine, in DS from 20 micrograms/liter. In PS I, major changes included proliferation of smooth endoplasmic reticulum, atypical mitochondria and lysosomes, as well as gradual alterations of the apical plasmalemma. Typical changes in PS II cells were a proliferation of peroxisomes and ring- and cup-shaped mitochondria, as well as alterations in the basal labyrinth; DS cells were characterized by a proliferation of atypical mitochondria with longitudinally oriented cristae, disorganization of Golgi fields and vacuolization of the cell base. Results document that different segments of the renal tubule in rainbow trout react not only with different levels of sensitivity to atrazine exposure, but also in a segment-specific way. Moreover, comparison of effects induced by atrazine with those resulting from chronic exposure to the herbicide linuron revealed a distinct substance specificity in the reaction of different tubular segments.
Ultrastructural alterations in liver and gills of embryonic and larval zebrafish (Danio rerio) following prolonged exposure to waterborne 0.05, 0.5, and 5 mg/L 4-chloroaniline for up to 31 days as well as after a 14-day regeneration period were investigated by means of light and electron microscopy. Acute toxicity was also tested at 25 and 50 mg/L. Survival of zebrafish embryos and larvae was only impaired from 25 mg/L 4-chloroaniline, but-after a transient stimulation following exposure to 0.5 mg/L-4-chloroaniline hatching was retarded after exposure to >/=5 mg/L, and fish displayed increasing rates of abnormal development and pigmentation. In contrast, hepatocytes displayed a time- and dose-dependent response from 0.05 mg/L 4-chloroaniline, including changes in nuclei, mitochondria, peroxisomes, endoplasmic reticulum, Golgi fields, lysosomes, and hepatic glycogen and lipid stores, as well as invasion of macrophages. In gills, dose-dependent effects were evident from 0.5 mg/L 4-chloroaniline and included deformation of secondary lamellae due to vacuolization and desquamation of respiratory epithelial cells in conjunction with dilation of intercellular spaces. Respiratory epithelial cells displayed progressive mitochondrial changes, induction of cytoplasmic myelinated structures, augmentation of lysosomes, and modifications of Golgi fields. Erythrocytes were severely deformed. A 14-day regeneration period was sufficient for almost complete recovery of pathological symptoms in both liver and gills. Only minor volumetric changes in hepatocellular organelles and a limited number of myelinated bodies, lysosomes, and cytoplasmic vacuoles were reminiscent of prior 4-chloroaniline exposure. In both qualitative and quantitative terms, most effects in hepatocytes after exposure of embryonic and larval zebrafish to waterborne 4-chloroaniline are comparable to the reaction of hepatocytes in adult zebrafish liver after prolonged sublethal exposure as well as in larval zebrafish after microinjection. Morphological changes in erythrocytes indicate disturbance of respiration as an additional mode of action of 4-chloroaniline.
Biochemical and ultrastructural changes in teleost liver following subacute ex-posure to PCB Disposition and metabolism of aniline in Fisher 344 rats and C57BL/6xC3H F1 mice
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Morphological and biochemical studies on liver, kidney, and gill of fishes affected by pesticides Growth and survival of developing steelhead trout (Salmo gairdneri) continu-ously or intermittently exposed to copper
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Develop-ment of newly formed nephrons in the goldfish kidney following hexachlorobutadiene-induced nephrotoxicity Renal tubular cell regeneration, cell proliferation, and chronic nephrotoxicity in the goldfish Carassius auratus following exposure to a single sublethal dose of hexachlor-obutadiene
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Analyse ultrastructurale des altérations hépatiques induites chez des poissons marins (Mugilidés) par un herbicide: l'atrazine (s-triazine)
- S Biagianti
- J Bruslé
Disposition and metabolism of aniline in Fisher 344 rats and C57BL/6xC3H F1 mice
- Mac Carthy
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Cytological alterations in fish fibrocytic R1 cells as an alternative test system for the detection of sublethal effects of environmental pollutants: A case-study with 4-chloroaniline
- T Zahn
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- T Braunbeck