The EYTEX™ System and the Neutral Red Uptake Inhibition Assay in Cultured Hep G2 Cells as Alternative Methods for In Vivo Eye Irritation Following the EEC Protocol
Paul J. Dierickx
Cellular and Molecular Targets of Benzo[a]pyrene and Metal Toxicity in Xenopus laevis Embryos and in Hep G2 Cells
Marina Camatini, Patrizia Bonfanti, Anita Colombo, Chiara Urani and Silvia Crippa
This paper describes the use of two in vitro systems (stage 35 Xenopus laevis embryos and the human hepatoblastoma cell line, Hep G2) to study effects of some environmental contaminants (benzo[a]pyrene, copper and zinc), which are representative of chemicals with different cell targets and mechanisms of action. The ability to activate benzo[a]pyrene and to metabolise it with the cytochrome P4501A isozyme were demonstrated in both in vitro systems by assessing the formation of water-soluble and protein-bound benzo[a]pyrene metabolites and by immunochemical analysis. In X. laevis embryos, the formation of DNA adducts demonstrated the ability to produce benzo[a]pyrene reactive metabolites. Moreover, in Hep G2 cells, the cytoskeletal protein, tubulin, and the reduced form of glutathione proved to be the cellular targets of copper and zinc toxicity. In response to metal-induced stress in Hep G2 cells, there was a cytoplasmic reorganisation of heat shock protein, Hsp 70. In conclusion, the in vitro systems provide for a rapid evaluation of heterogeneous compounds such as benzo[a]pyrene and heavy metals that differ in toxic potency and mechanisms of action. They could also be used to study the mechanisms of toxic action and to identify specific cellular and molecular targets.
Prediction of Human Acute Toxicity by the Hep G2/24-hour/Total Protein Assay, with Protein Measurement by the CBQCA Method
Paul J. Dierickx
In our previously described Hep G2/24-hour/total protein assay, protein levels were measured by using the Lowry method. This assay was the best acute in vitro assay for the prediction of human toxicity within the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) study. In order to increase the MEIC data-base with a wider range of chemicals, we were interested in introducing the more practical 3-(4-carboxybenzoyl)-quinoline-2-carboxaldehyde (CBQCA) method for the quantification of the total protein content. Therefore, we investigated whether the same good results for the prediction of acute human toxicity would be obtained with the CBQCA method. The cells were treated for 24 hours, then cytotoxicity was determined by measuring the total protein content with CBQCA. The results were quantified by using the PI50c: the concentration (in mM) of test compound required to reduce the total protein content measured with the CBQCA-method by 50% as compared to the control cells. The results were compared with the PI50, the corresponding value when the Lowry method was used. A relatively low correlation was observed between PI50 and PI50c, reflecting the large and unexpected, differences when using the two protein assays. However, when comparing the log PI50c with the human toxicity, a correlation coefficient of r² = 0.761 (n = 44) was obtained for exactly the same series of MEIC chemicals. This value is clearly higher than that for the Lowry method (r² = 0.695). Compared to the Lowry method originally used, the Hep G2/24-hour/CBQCA total protein assay has the additional important advantage that it can be very easily adapted for large-scale analyses with robotic systems, including the on-line calculation of the results.