Acute and subacute toxic effects produced by microcystin-YR on the fish cell lines RTG-2 and PLHC-1.

Approximately 80 microcystins (MCs) variants have been isolated in surface water worldwide. The toxicity of the most frequently MCs are encountered, MC-LR and MC-RR, has been extensively studied in humans and animals. However, studies dealing with MC-YR toxicity are still scarce. In this work, the toxic effects of MC-YR were investigated in the fish cell line PLHC-1, derived from a hepatocellular carcinoma of the topminnow Poeciliopsis lucida, and RTG-2 fibroblast-like cells derived from the gonads of rainbow trout Oncorhynchus mykiss. After 48 h, morphological and biochemical changes (total protein content, neutral red uptake and methylthiazol tetrazolium salt metabolization) were determined. The most sensitive endpoint for both cell lines was the reduction of total protein content, with EC(50) values of 35 microM for PLHC-1 cells and 67 microM for the RTG-2 cell line. Lysosomal function and methylthiazol tetrazolium salt metabolization were stimulated at low concentrations, while they decreased at high doses. Increase of piknotic cells, rounding effects, reduction in cell number and cell size, hydropic degeneration, and death mainly by necrosis but also by apoptosis were observed in the morphological study. Furthermore, PLHC-1 cells are more sensitive than RTG-2 cells to MC-YR exposure. These results were similar to those obtained when both cell lines were exposed for 24h to a Microcystis aeruginosa isolated strain extract containing MC-LR.

The use of the fish cell lines RTG-2 and PLHC-1 to compare the toxic effects produced by microcystins LR and RR.

Cyanobacterial toxins, especially microcystins (MC), are found in eutrophied waters through the world. Acute poisonings of animals and humans has been reported following MC exposure. In the present study, two fish cell lines, PLHC-1 and RTG-2, were evaluated after exposure to the cyanobacterial toxins MC-LR and MC-RR. The effects of different concentrations of the toxins were investigated in both cell lines at morphological and biochemical levels (total protein content, lactate dehydrogenase leakage, lysosomal activity and succinate dehydrogenase activity). The results obtained showed a decrease in protein content and no relevant increase in cell disruption, except for MC-LR in PLHC-1 cells. Morphological changes produced by microcystins were cellular swelling, blebbling, rounding, reduction in the cell number and increase in the number and size of lysosomal bodies. In addition, steatosis was produced in hepatoma PLHC-1 cells, particularly with MC-RR. Furthermore, the fish PLHC-1 cell line was more sensitive than RTG-2 cells to the cyanobacterial toxins compared, being the stimulation of the lysosomal function and the induction of steatosis the most specific changes detected.

Ecotoxicological evaluation of diethanolamine using a battery of microbiotests.

In order to investigate the potential ecotoxicity of diethanolamine (DEA), a battery of model systems was developed. DEA is widely used as a chemical intermediate and as a surface-active agent in cosmetic formulations, pharmaceuticals and agricultural products. DEA was studied using ecotoxicological model systems, representing four trophic levels, with several bioindicators evaluated at different exposure time periods. The battery included bioluminescence inhibition of the bacterium Vibrio fischeri, growth inhibition of the alga Chlorella vulgaris and immobilization of the cladoceran Daphnia magna. Cell morphology, total protein content, neutral red uptake, MTS metabolization, lysosomal function, succinate dehydrogenase activity, G6PDH activity, metallothionein levels and EROD activity were studied in the hepatoma fish cell line PLHC-1, derived from Poeciliopsis lucida. The systems most sensitive to DEA were both D. magna and V. fischeri, followed by C. vulgaris and the fish cell line PLHC-1. The most prominent morphological effect observed in PLHC-1 cultures exposed to DEA was the induction of a marked steatosis, followed by death at high concentrations, in some cases by apoptosis. The main biochemical modification was a nearly three-fold increase in metallothionein levels, followed by the stimulations of lysosomal function and succinate dehydrogenase and G6PDH activities. Judging by the EC(50) values in the assay systems, DEA is not expected to produce acute toxic effects in the aquatic biota. However, chronic and synergistic effects with other chemicals cannot be excluded.