Comparable metabolite patterns of benzo[a]pyrene and cinnamic aldehyde in human skin ex vivo and human skin models in vitro indicate comparable metabolic capacities

Alemán. In vitro skin models are widely recommended in toxicological test guidelines for addressing penetration, as well as corrosionand irritation. However, the suitability of these test systems for other toxicological endpoints, such as genotoxicity or skin sensitization depends on their metabolic competence to transformnon-toxic parental compounds into relevant metabolites of toxicological concern. Initial studies on metabolizing enzymes in skin models have focused on expression patterns of cytochrome P450 (CYP) monooxygenases and selected detoxifying phaseII enzymes. Besides gene expression, comparatively little is known about enzyme activities present in skin models. Both CYP1A1 and 1B1 are thought to initiate the biotransformation of benzo[a]pyrene (BP), thereby resulting in a complex pattern of metabolites due to various parallel or alternate pathways of BP conversion. We have analyzed the induction of CYP1 enzymes, as well as CYP activities in full thickness models, showing that these enzymes are preferentially expressed within the epidermal layers. In order to address the metabolic capacity directly, we have compared the metabolite patterns in BP-exposed 3D skin models, excised human skin and cultured primary human keratinocytes. Importantly, the patterns of eight BP-metabolites detected, including BP-r-7,t-8,t-9,c-10-tetrahydrotetraol, 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 were comparable between human skin and the MatTek epidermal and full thickness skin models. All analyzed BP metabolites were also detectedin primary human keratinocytes. However, these primary cultures appeared to metabolize BP at lower rates than human skin. Further, we also compared the metabolic transformation of cinnamic aldehyde into cinnamic acid and cinnamic alcohol in the skin models and human skin. Our results confirm a sufficient metabolic competence of the selected skin models and support their applicability for replacing in vivo tests for genotoxicity and other endpoints that rely on metabolism. The BfR coordinates a German multi-center project (BMBF Funding ID 0315226 A-D) aimed at developing and prevalidating the Comet assay in skinmodels. Initial observations also confirm that pro-mutagens are converted into their active forms, which can be detected in this assay.