Paul J. Dierickx

Correlation between the in vitro cytotoxicity to cultured fathead minnow fish cells and fish lethality data for 50 chemicals

Abstract Neutral red uptake inhibition was investigated as an alternative method for the assessment of ecotoxicity. The 50 chemicals tested include different kinds of inorganic compounds and organic compounds such as alcohols, acids, ketones, esters, and others. The xenobiotics were applied at different concentrations to cultured FHM (fathead minnow fish) cells. After 2 hours contact time neutral red uptake inhibition was measured. The results are expressed as the NI50 value, which is the concentration of test compound required to induce a 50% reduction in neutral red uptake. The NI50 values were compared with fish lethality data (LC50) obtained in golden orfe by Juhnke and Ludemann (1978). A false positive cytotoxic result was observed for 1,4-dioxane. For the remainder 49 chemicals a correlation coefficient r=0.89 was found. Hence it appears that the neutral red uptake inhibition in cultured fish cells can be considered as a valuable tool for in vitro ecotoxicity testing of a wide variety of chemicals.

Glutathione S-transferases of Aulacorthum solani and Acyrthosiphon pisum: partial purification and characterization

Glutathione S-transferases (GST) play an important role in the detoxification of many substances including allelochemicals from plants. Brassicaceae plants contain glucosinolates and emit volatile isothiocyanates which affect the GST system. A comparison of the GST of two aphid species, the generalist Aulacorthum solani found on Brassicaceae and the Fabaceae specialist Acyrthosiphon pisum, was made to try to explain their respective feeding behaviour. Differences of GST were determined among the two aphid species based on purification by affinity chromatography, SDS-PAGE and on kinetic studies. Purification yields using an epoxy-activated Sepharose 6B column were highly different for the two aphid species (18% and 34% for A. solani and A. pisum, respectively). These variations were confirmed by SDS-PAGE. While only a 27-kDa band was observed for A. pisum, two bands of approximately 25-kDa were visualized for the generalist aphid, A. solani. Considering the kinetic results, differences of Km and Vmax were observed following the aphid species when a range of substrates (CDNB and DCNB) and GSH concentrations were tested. Studies on the detoxification enzymes of generalist and specialist herbivores would be undertaken to determine accurately the effect of the host plant on the organisms eating them, particularly in terms of biochemical and ecological advantages.

Effects of foetal calf serum on cell viability, cytotoxicity and detoxification in the two kidney-derived cell lines LLC-PK1 and MDCK

Foetal calf serum (FCS) dependent cell viability, cytotoxicity and detoxification were investigated in MDCK and LLC-PK1 cells. FCS was used at 0-10% (v/v). Viability and cytotoxicity were measured by neutral red uptake and by the MTT test. Viability of LLC-PK1 was strongly dependent on the FCS concentration, but that of MDCK cells only to a very limited extent. For both cell lines the cytotoxicity of HgCl(2) was FCS concentration dependent: lower toxicity was observed with 5-10% FCS than with 0-1% FCS. This effect was not observed for paracetamol. The results could not be explained by altered glutathione or glutathione S-transferase. The optimal FCS concentration of 1%, necessary to retain cell viability, had a limited influence on cytotoxicity. FCS concentration must be taken into consideration when cytotoxicity data from different studies are compared.

Increased cytotoxic sensitivity of cultured FHM fish cells by simultaneous treatment with sodium dodecyl sulfate and buthionine sulfoximine

A previously described neutral red uptake inhibition assay on cultured fathead minnow (FHM) fish cells revealed a good correlation with fish toxicity data (Brandao et al., 1992) for a series of 50 test compounds, belonging to completely different chemical classes. The major drawback was the lower sensitivity of the cytotoxicity assay. Aiming at a higher sensitivity the assay was adapted by reducing the cell number, by a longer treatment period, and by simultaneous treatment with sodium dodecyl sulfate and buthionine sulfoximine (BSO). The fluorimetrically measured protein content was chosen as the endpoint. The endogenous glutathione (GSH) content was reduced by 54% of the test chemicals, confirming the importance of GSH in detoxification processes. Higher sensitivity was, at least partly, obtained by treating the FHM cells with BSO, reducing the GSH content to 22% of that in control cells. The cytotoxicity of the 50 chemicals was measured using the modified more sensitive assay. Although some exceptions were observed, this new assay is at least one order of magnitude more sensitive. Toxicity values comparable with fish toxicity data were obtained, making the assay sensitive enough to measure the toxicity of environmental water samples.

Comparison between fish lethality data and the in vitro cytotoxicity of lipophilic solvents to cultured fish cells in a two-compartment model

Abstract Cytotoxicity testing of non-hydrosoluble chemicals offers a major problem because cells are always cultured in aqueous media. An adaptation of the two-compartment model of Boue-Grabot et al. (1992) is reported here. The cytotoxicity of 19 lipophilic solvents was measured on cultured FHM (fathead minnow fish) cells. The FHM cells were seeded in transwells on a 0.4 μm pore membrane (upper compartment) which are placed in the wells of a 24 well culture plate (lower compartment). The transwells were then placed in wells containing the test chemical, solubilized in paraffin. After 24h the total protein content was measured. The relative toxicity is expressed by the EC50. This is the concentration of test chemical in the lower compartment required to induce a 50% inhibition of the total protein content in the upper compartment. No linear correlation was obtained between the EC50 of the lipophilic solvents and the in vivo fish lethality data obtained in golden orfe by Juhnke and Ludemann (1978). Nevertheless, this method allows the ranking of quantitative cytotoxicity data of lipophilic chemicals towards cultured fish cells.

The cytosolic glutathione S-transferase isoenzymes in the dog kidney cortex as compared with the corresponding MDCK renal cell line.

Cytosolic glutathione S-transferase (GST) (EC 2.5.1.18) isoenzymes of dog kidney and MDCK (an established dog renal cell line) were purified and studied. Specific GST activity was 248 and 317 nmol/min/mg protein, for dog and MDCK, respectively. Cytosolic GST was only partially purified by glutathione affinity chromatography, a substantial amount (43% and 84% for dog kidney and MDCK, respectively) of the GST activity was found in the flow-through fraction. Affinity bound GST was separated into 6 and 3 isoenzymes by anionic chromatofocusing for dog and MDCK, respectively. Flow-through GST was purified by gel filtration, anion exchange chromatography and anionic chromatofocusing showing only one GST isoenzyme, with distinct features from the affinity bound GST, for both dog and MDCK. The isoenzymes were characterized by their kinetic properties, subunit composition, specific substrates and inhibitors and immunoblot. The major dog GSTs (DII, DIV and DVI) correspond to the MDCK isoenzymes (MI, MII and MIII). Comparable pI values, a comparable affinity towards GSH and comparable sensitivities towards the inhibitors N-ethylmaleimide (NEM), triphenyltin chloride, cibacron blue and hematin were observed for the corresponding isoenzymes: DII and MI, DIV and MII, DVI and MIII. Co-electrophoresis showed that the subunit composition was identical for DII and MI, and for DIV and MII. Inhibitor and substrate sensitivities showed that the affinity bound GSTs belong to class pi and mu, the presence of class pi was confirmed by immunoblot analysis. One homodimeric GST isoenzyme was observed in the dog kidney and MDCK flow-through. Both dog and MDCK isoenzyme have a nearly neutral pI, a high affinity towards CDNB and an equal sensitivity towards triphenyltin chloride, cibacron blue and hematin. However, based on inhibitor studies and immunoblot, this isoenzyme could not be attributed to an identified GST class. The overall isoenzyme pattern of dog and MDCK affinity bound and flow through GST is comparable. The dog and MDCK affinity bound GSTs have similar characteristics and all belong to class mu or pi.

Correlation between fish lethality data and the cytotoxicity to cultured fish cells of lipophilic solvents emulsified by ultrasonication

Abstract Cytotoxicity testing of non-hydrosoluble chemicals poses a major problem because cells are always cultured in aqueous media. The test chemicals were, therefore, emulsified by ultrasonication, before treating the cells for 2h with different concentrations. The cytotoxicity of 19 lipophilic solvents, belonging to structurally unreleated chemical classes, was measured on cultured FHM (fathead minnow fish) cells by the neutral red uptake inhibition assay. The results are expressed as the NI50, which is the concentration of test compound required to induce a 50% reduction in neutral red uptake. A correlation coefficient r=0.86 was obtained when the NI50 values were compared with fish lethality data (LC50) obtained on golden orfe by Juhnke and Ludemann (1978). Hence it appears that emulsification of lipophilic chemicals by ultrasonication allows to measure their cytotoxicity on cultured cells, and that this method can be considered as a valuable alternative tool for fish lethality testing.

Characterization of the major cytosolic glutathione transferase in two monkey kidney derived cell lines.

Glutathione transferases (GST) are multifunctional phase II enzymes. They play an important role in the biotransformation and detoxification of xenobiotics (Mannervik and Danielson, 1988). Their multiplicity is assumed to result from the need to conjugate numerous types of substances differing in the nature of their electrophilic centre and their molecular structure. In human kidney the GST activity is nearly of the same level as in the liver (Pacifici et aI., 1988). Here we report the characterization of the major cytosolic GST in two established kidney cell lines Vero cells (from African green monkey) and LLC-MK2 (from Rhesus monkey).

Isolation and characterisation of the class alpha, mu and pi glutathione transferases in LLC-PK1 and pig kidney

Glutathione S-transferase (GST) isoenzymes from pig kidney cortex and LLC-PK1 (an established cell line derived from the pig proximal tubule) were purified by affinity chromatography, anionic and cationic chromatofocusing. Purification revealed nine isoenzymes in the pig kidney cortex and five isoenzymes in the LLC-PK1 cell line. SDS-polyacrylamide gel electrophoresis showed that the pig kidney cortex isoenzymes were homo- or heterodimeric; LLC-PK1 isoenzymes, however, were homodimeric. Isoenzymes from pig and LLC-PK1 showed a higher affinity towards glutathione. The isoenzymes were further characterised and divided into the different GST classes by studying specific inhibitors, specific substrates and immunological properties. Pig GSTs belong to class alpha, mu and pi. The GSTs in LLC-PK1 cells, on the other hand, belong to class pi and mu. The isoenzyme pattern in LLC-PK1 cells indicates the dedifferentiation of this particular cell line compared with the pig kidney cortex.