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 to environmental risk assessment for chemicals and chemical mixtures.
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.
Zahn
T
T
Department of Zoology I, University of Heidelberg.
Hauck
C
C
Holzschuh
J
J
Braunbeck
T
T
D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D002478:Cells, Cultured; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008099:Liver / Q000166:cytology* / Q000473:pathology / Q000648:ultrastructure; D008854:Microscopy, Electron; D017686:Oncorhynchus mykiss; D012722:Sewage; D014865:Waste Disposal, Fluid; D014873:Water Pollutants / Q000633:toxicity*
D012722:Sewage; D014873:Water Pollutants; D007770:L-Lactate Dehydrogenase