Abstract
CYP enzyme induction is a sensitive biomarker for phenotypic metabolic competence of in vitro test systems; it is a key event associated with thyroid disruption, and a biomarker for toxicologically relevant nuclear receptor-mediated pathways. This paper summarises the results of a multi-laboratory validation study of two in vitro methods that assess the potential of chemicals to induce cytochrome P450 (CYP) enzyme activity, in particular CYP1A2, CYP2B6, and CYP3A4. The methods are based on the use of cryopreserved primary human hepatocytes (PHH) and human HepaRG cells.The validation study was coordinated by the European Union Reference Laboratory for Alternatives to Animal Testing of the European Commission's Joint Research Centre and involved a ring trial among six laboratories. The reproducibility was assessed within and between laboratories using a validation set of 13 selected chemicals (known human inducers and non-inducers) tested under blind conditions. The ability of the two methods to predict human CYP induction potential was assessed. Chemical space analysis confirmed that the selected chemicals are broadly representative of a diverse range of chemicals.The two methods were found to be reliable and relevant in vitro tools for the assessment of human CYP induction, with the HepaRG method being better suited for routine testing. Recommendations for the practical application of the two methods are proposed.
Highlights
The toxicity profile of an exogenous chemical to which the body is exposed depends on the toxicity of the parent compound, and on any toxicologically relevant metabolites that may be formed during metabolism and on the xenobiotic's ability to induce biotransformation enzymes that affect its rate of metabolism (Tsaioun et al, 2016)
For all three cytochrome P450 (CYP) enzymes, a consistently higher reproducibility for both BBRL and BLRB was obtained for HepaRG cells compared to primary human hepatocytes (PHH)
For HepaRG cells BBRL values are similar for a given batch except in the case of CYP1A2
Summary
The toxicity profile of an exogenous chemical (xenobiotic) to which the body is exposed depends on the toxicity of the parent compound, and on any toxicologically relevant metabolites that may be formed during metabolism and on the xenobiotic's ability to induce biotransformation enzymes that affect its rate of metabolism (Tsaioun et al, 2016). Over the last two decades, considerable progress has been made in developing in vitro metabolism methods based on human test systems (Coecke et al, 2013; Donato et al, 2008; Vermeir et al, 2005; Vinken and Hengstler, 2018). There were no formally validated test systems based on intact functional human hepatic cells capable of maintaining key metabolic activity functions for up to 3 days in culture. Of all xenobiotic-metabolising enzymes, the Cytochrome (CYP) P450 enzymes are of particular importance due to their abundance and functional versatility (Raunio et al, 2015). They may transform a xenobiotic into a harmless metabolite (detoxification) or, vice versa, a nontoxic parent compound into a toxic metabolite.
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