COMPARATIVE STUDIES ON THE BIOTRANSFORMATION OF BENZO[A]PYRENE IN HUMAN KERATINOCYTES, IN VITRO SKIN MODELS, AND SKIN EX VIVO: METABOLITE PATTERNS AS INDICATOR FOR THE METABOLIC CAPACITY OF SKIN EQUIVALENTS.
In vitro skin models are increasingly important for toxicological testing strategies to facilitate risk assessment and classification of chemicals. The reliability of these tests depends on the capacity of skin equivalents to metabolize xenobiotics in a similar way as found for human skin. So far, studies aimed at characterizing the metabolic competence of in vitro models have concentrated on analyses of expression patterns and activities of selected cytochrome P450-dependent monooxygenases (CYP) and various phase II enzymes. However, uncertainties remain whether expression of mRNA transcripts or in vitro activities of enzymes are sufficient and reliable enough to compare between the metabolic capacities of skin models and human skin. As an alternative approach, the Federal Institute for Risk Assessment participates in a multi-centered research project funded by the Federal Ministry of Education and Research (BMBF). This project aims to determine and to compare metabolite profiles obtained from a range of different skin models upon exposure to certain test compounds. We selected benzo[a]pyrene (BP), because alternate enzymatic pathways apply for this substance that result in a complex profile of metabolites. We established exposure models, homogenization and extraction protocols, as well as analytical methods to detect a wide spectrum of BP metabolites, including BP-r-7,t-8,t-9,c-10-tetrathydrotetrol, trans-BP-7,8-dihydrodiol, trans-BP-9,10-dihydrodiol, BP-1,6-dione, BP-7,8-dione, 3-OH-BP, 7-OH-BP and 8-OH-BP. Our data show that the overall metabolite profiles obtained from different skin models can serve as valuable tool to assess the metabolic capacity of in vitro skin models and their comparability with human skin ex vivo. Along with other test substances and additional enzymatic assays, the metabolic profiles will allow for identification and definition of an optimal metabolic capacity to be expected for skin models that successfully mimic the corresponding activity in human skin in vivo and thus may serve as a suitable starting material for developing novel testing strategies. A central objective of this project is the adaption of skin models to toxicological endpoints that depend on appropriate biotransformation, including genotoxicity and sensitization. We also discuss our initial work to adapt the COMET assay to the special requirements of tissue equivalents.
3-OH-BP, 7-OH-BP, 8-OH-BP, Anti-BP-7,8-dihydrodiol-9-10-epoxide, Benzo[a]pyrene metabolites, BP-1,6 dione, BP-7,8 dione, BP-r-8,t-8,t-9,c-10-tetrahydrotetrol, Cytochrome P450, Dermal biotransformation, EpiDerm-200, EpiDermFT-400, Ex vivo skin, Metabolic capacity, Phase II enzymes, Trans-BP-7,8-dihydrodiol, Trans-BP-9,10-dihydrodiol
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