ABSTRACT
Chemicals often require metabolic activation to become genotoxic. Established test guidelines recommend the use of the rat liver S9 fraction or microsomes to introduce metabolic competence to in vitro cell-based bioassays, but the use of animal-derived components in cell culture raises ethical concerns and may lead to quality issues and reproducibility problems. The aim of the present study was to compare the metabolic activation of cyclophosphamide (CPA) and benzo[a]pyrene (BaP) by induced rat liver microsomes and an abiotic cytochrome P450 (CYP) enzyme based on a biomimetic porphyrine catalyst. For the detection of genotoxic effects, the chemicals were tested in a reporter gene assay targeting the activation of the cellular tumor protein p53. Both chemicals were metabolized by the abiotic CYP enzyme and the microsomes. CPA showed no activation of p53 and low cytotoxicity without metabolic activation, but strong activation of p53 and increased cytotoxicity upon incubation with liver microsomes or abiotic CYP enzyme. The effect concentration causing a 1.5-fold induction of p53 activation was very similar with both metabolization systems (within a factor of 1.5), indicating that genotoxic metabolites were formed at comparable concentrations. BaP also showed low cytotoxicity and no p53 activation without metabolic activation. The activation of p53 was detected for BaP upon incubation with active and inactive microsomes at similar concentrations, indicating experimental artifacts caused by the microsomes or NADPH. The activation of BaP with the abiotic CYP enzyme increased the cytotoxicity of BaP by a factor of 8, but no activation of p53 was detected. The results indicate that abiotic CYP enzymes may present an alternative to rat liver S9 fraction or microsomes for the metabolic activation of test chemicals, which are completely free of animal-derived components. However, an amendment of existing test guidelines would require testing of more chemicals and genotoxicity end points.
ABSTRACT
High-throughput cell-based bioassays are used for chemical screening and risk assessment. Chemical transformation processes caused by abiotic degradation or metabolization can reduce the chemical concentration or, in some cases, lead to the formation of more toxic transformation products. Unaccounted loss processes may falsify the bioassay results. Capturing the formation and effects of transformation products is important for relating the in vitro effects to in vivo. Reporter gene cell lines are believed to have low metabolic activity, but inducibility of cytochrome P450 (CYP) enzymes has been reported. Baseline toxicity is the minimal toxicity a chemical can have and is caused by the incorporation of the chemical into cell membranes. In the present study, we improved an existing baseline toxicity model based on a newly defined critical membrane burden derived from freely dissolved effect concentrations, which are directly related to the membrane concentration. Experimental effect concentrations of 94 chemicals in three bioassays (AREc32, ARE-bla and GR-bla) were compared with baseline toxicity by calculating the toxic ratio (TR). CYP activities of all cell lines were determined by using fluorescence-based assays. Only ARE-bla showed a low basal CYP activity and inducibility and AREc32 showed a low inducibility. Overall cytotoxicity was similar in all three assays despite the different metabolic activities indicating that chemical metabolism is not relevant for the cytotoxicity of the tested chemicals in these assays. Up to 28 chemicals showed specific cytotoxicity with TR > 10 in the bioassays, but baseline toxicity could explain the effects of the majority of the remaining chemicals. Seven chemicals showed TR < 0.1 indicating inaccurate physicochemical properties or experimental artifacts like chemical precipitation, volatilization, degradation, or other loss processes during the in vitro bioassay. The new baseline model can be used not only to identify specific cytotoxicity mechanisms but also to identify potential problems in the experimental performance or evaluation of the bioassay and thus improve the quality of the bioassay data.
