WP6: Improving in vitro/in vivo correlations – Metabolism

 

WP6 results, download the entire summary: Results WP6.pdf

 

The objective of WP6 has been to evaluate if the metabolism may influence the toxicity of a compound, since it may actually be a metabolite (a substance produced by biotransformation of the parent chemical in the liver) that is responsible for the observed toxicity.

 

The work in WP6 has been divided into three parts:

1. Simple models for metabolism-dependent toxicity
2. New strategies to incorporate metabolic capabilities into cell lines
3. Computer-based prediction of metabolism and integration of metabolism data into toxicity screening

 

1. Simple models for metabolism-dependent toxicity

In order to evaluate if toxicity is dependent on metabolism, the cytotoxic effects of the 57  ACuteTox reference chemicals, as well as 5 additional bioactivable compounds, have been compared between a metabolic component model (primary hepatocytes) and a non-metabolising cell type (HepG2) by the use of MTT assay.

 

By comparing the concentration-toxicity curves of each compound in both models it is possible to ascertain whether the molecule elicits toxic effects preferentially on hepatocytes suggesting that a bioactivation of the xenobiotic is required. A number of compounds that requires bioactivation in order to be toxic were identified.

 

To examine the robustness of the strategy, intra-assay, inter-assay as well as intra-laboratory variability was investigated for each cell system. A low variability (CV<10%), both intra-plate and intra-assay, was obtained in all laboratories, however, the intra laboratory variability needs to be improved.

 

The Phototox software has been used to develop a software that fit the needs of the ACuteTox project, such as comparing dose-response curves from three different cell cultures. A first version of the software was developed during 2008. This will enable better comparisons of IC50 between compounds.

 

2. New strategies to incorporate metabolic capabilities into cell lines

HepG2 cells have been transfected with recombinant-defective adenoviral vectors encoding for the major CYP genes involved in foreign compound metabolism. The method has been adapted to 96-well plates in order to function as a screening assay.

 

3. Computer-based prediction of metabolism and integration of metabolism data into toxicity screening

Another aim was to investigate how METEOR and DEREK software performs in prediction of metabolic fate of compounds with known biotransformation. Fourteen ACuteTox reference compounds were tested, indicating that METEOR appears to be an interesting alternative for in silico prediction of metabolism. Furthermore, it was concluded that even if the DEREK programme does not predict acute toxicity per se, important information regarding the toxic profile of the tested substances can be gained.