Acute and sublethal toxicity of seepage waters from garbage dumps to permanent cell lines and primary cultures of hepatocytes from rainbow trout (Oncorhynchus mykiss) : a novel approach risk assessment for chemicals and chemical mixtures
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In order to evaluate the suitability of cytopathological criteria in isolated fish hepatocytes as endpoints in (eco)toxicological research, liver cells isolated from rainbow trout (Oncorhynchus mykiss) by collagenase perfusion were exposed in vitro for up to 5 days to sublethal dilutions of two seepage water samples collected from garbage dumps. Hepatocytes were analysed with respect to acute (lactate dehydrogenase leakage) and sublethal toxicity (electron microscopy, stereology). In addition, acute toxicity (24 h) was tested in the piscine fibrocytic cell line R1 by means of crystal violet staining and neutral red retention. Acute toxicity in R1 cells and isolated hepatocytes could only be documented for sample I at dilutions of 1:2 and 1:4. This difference in toxicity could be corroborated by cytological alterations in isolated hepatocytes, which could be documented for dilutions of 1:100 and 1:8 in samples I and II, respectively. Ultrastructural changes were time- and dose-dependent and included reduction of hepatocellular volume, disturbance of intracellular compartmentation, modified heterochromatin distribution, transformation of rough endoplasmic reticulum into concentric membrane whorls, proliferation of lysosomes and cytoplasmic vacuoles, as well as reduction of hepatocellular glycogen. Although several hepatocellular reactions were found after exposure to either sample, the syndrome of ultrastructural alterations allowed clear differentiation between the two samples. Results illustrate that cytological effects far below macroscopically detectable damage can be discovered not only in intact fish, but also in fish cell culture systems. On the basis of the data presented, a multi-tiered test procedure for aquatic toxicity assessment exclusively based on tests with fish cell culture systems is proposed: (1) rapid screening for acute toxicity with permanent cell lines; (2) short-term tests with more complex, yet more sensitive systems such as primary hepatocytes with straightforward biochemical endpoints; (3) prolonged exposure of isolated hepatocytes in combination with ultrastructural and biochemical investigations as a sensitive tool to detect adverse effects at environmentally relevant toxicant concentrations.
Zusammenfassung in einer weiteren Sprache
Um die Eignung cytopathologischer Veränderungen in isolierten Fischhepatocyten als Endpunkte in (öko-)toxikologischen Studien zu untersuchen, wurden Leberzellen aus der Regenbogenforelle (Oncorhynchus mykiss) durch Collagenaseperfusion isoliert und in vitro bis zu 5 Tage subletalen Verdünnungen von zwei Sickerwässern exponiert. Die Hepatocyten wurden auf akute (Laktatdehydrogenasefreisetzung) und subletale Toxizität (Elektronenmikroskopie) geprüft. Zusätzlich wurde die akute Toxizität in der fibrocytenähnlichen Fischzellinie R 1 mit Hilfe von Kristallviolettfärbung und Neutralrotretention bestimmt. Eine akut toxische Wirkung konnte in beiden Zelltypen nur für die erste Probe in Verdünnungen von 1:2 bzw. 1:4 belegt werden. Dieser Unterschied in der Toxizität von Probe I und II ließ sich mit cytologischen Untersuchungen an isolierten Hepatocyten für Verdünnungen von 1:100 bzw. 1:8 bestätigen. Die zeit- und dosisabhängigen ultrastrukturellen Veränderungen umfassten Verringerung der Zellgröße, Störung der intrazellulären Kompartimentierung, veränderte Heterochromatinverteilungsmuster, Umbau des rauhen endoplasmatischen Retikulumus in konzentrische Membranwirbel, Proliferation von Lysosomen und Vakuolen im Cytoplasma sowie eine Abnahme an Glykogen. Obwohl mehrere Effekte nach Belastung mit beiden Proben nachzuweisen waren, ist eine Differenzierung der Proben anhand ultrastruktureller Veränderungen möglich. Die Ergebnisse belegen, dass cytologische Effkete lange vor makroskopischer Manifestation nicht nur im intakten Fisch, sondern auch in Zellkultursystemen aus Fischen nachweisbar sind. Auf der Basis der Befunde wird ein mehrstufiges Prüfschema für Toxizität von Wasserproben vorgeschlagen, das ausschließlich auf der Verwendung von Fischzellen beruht: (1) rasches Screening auf akute Toxizität mit permanenten Zellinien; (2) Kurzzeittests mit komplexeren, aber empfindlicheren Systemen wie isolierten Hepatocyten und cytologischen und biochemischen Endpunkten als hochempfindliches Werkzeug, um Schadstoffeffekte in umweltrelevanten Konzentrationen nachzuweisen.
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ZAHN, Thomas, Christof R. HAUCK, Jochen HOLZSCHUH, Thomas BRAUNBECK, 1995. Acute and sublethal toxicity of seepage waters from garbage dumps to permanent cell lines and primary cultures of hepatocytes from rainbow trout (Oncorhynchus mykiss) : a novel approach risk assessment for chemicals and chemical mixtures. In: Zentralblatt für Hygiene und Umweltmedizin. 1995, 196, pp. 455-479BibTex
@article{Zahn1995Acute-7363,
year={1995},
title={Acute and sublethal toxicity of seepage waters from garbage dumps to permanent cell lines and primary cultures of hepatocytes from rainbow trout (Oncorhynchus mykiss) : a novel approach risk assessment for chemicals and chemical mixtures},
volume={196},
journal={Zentralblatt für Hygiene und Umweltmedizin},
pages={455--479},
author={Zahn, Thomas and Hauck, Christof R. and Holzschuh, Jochen and Braunbeck, Thomas}
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<dcterms:abstract xml:lang="eng">In order to evaluate the suitability of cytopathological criteria in isolated fish hepatocytes as endpoints in (eco)toxicological research, liver cells isolated from rainbow trout (Oncorhynchus mykiss) by collagenase perfusion were exposed in vitro for up to 5 days to sublethal dilutions of two seepage water samples collected from garbage dumps. Hepatocytes were analysed with respect to acute (lactate dehydrogenase leakage) and sublethal toxicity (electron microscopy, stereology). In addition, acute toxicity (24 h) was tested in the piscine fibrocytic cell line R1 by means of crystal violet staining and neutral red retention. Acute toxicity in R1 cells and isolated hepatocytes could only be documented for sample I at dilutions of 1:2 and 1:4. This difference in toxicity could be corroborated by cytological alterations in isolated hepatocytes, which could be documented for dilutions of 1:100 and 1:8 in samples I and II, respectively. Ultrastructural changes were time- and dose-dependent and included reduction of hepatocellular volume, disturbance of intracellular compartmentation, modified heterochromatin distribution, transformation of rough endoplasmic reticulum into concentric membrane whorls, proliferation of lysosomes and cytoplasmic vacuoles, as well as reduction of hepatocellular glycogen. Although several hepatocellular reactions were found after exposure to either sample, the syndrome of ultrastructural alterations allowed clear differentiation between the two samples. Results illustrate that cytological effects far below macroscopically detectable damage can be discovered not only in intact fish, but also in fish cell culture systems. On the basis of the data presented, a multi-tiered test procedure for aquatic toxicity assessment exclusively based on tests with fish cell culture systems is proposed: (1) rapid screening for acute toxicity with permanent cell lines; (2) short-term tests with more complex, yet more sensitive systems such as primary hepatocytes with straightforward biochemical endpoints; (3) prolonged exposure of isolated hepatocytes in combination with ultrastructural and biochemical investigations as a sensitive tool to detect adverse effects at environmentally relevant toxicant concentrations.</dcterms:abstract>
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