EVALUATION OF IN VITRO MODELS FOR REACH TOXICITY TESTING: INDUCIBILITY OF XENOBIOTIC METABOLIZING ENZYME (XME) ACTIVITY IN IN VITRO HUMAN AIRWAY (EPIAIRWAY™) AND EPIDERMAL (EPIDERM™) MODELS.

Dermal and airway epithelia contain XMEs including phase I (oxidative) and phase II (conjugative) activities. These XMEs may play a role in biotransformation of drugs and chemicals, leading to altered drug activity or formation of toxic/mutagenic metabolites. Consideration of metabolic activity is therefore important for development of in vitro toxicity/genotoxicty assays required by REACH and similar testing programs. Baseline XME activity of dermal and airway epithelia is reportedly low, however many XMEs may be inducible via various nuclear receptor pathways. To evaluate inducibility of XME, in vitro human epidermal (EpiDerm™) and airway epithelial (EpiAirway™) models were exposed to a chemical cocktail including 3-methylcholanthrene (AhR activator), Rifampicin (CAR/PXR activator), Clofibric acid (PPAR activator), Chlorpromazine (CAR activator), and T-butylhydroquinone (ARE/EpRE activator). Induction of XME expression was evaluated by qPCR arrays containing 168 Phase I/II XME genes. The airway model expressed higher levels of many XMEs. Remarkably, CYP1A1 was induced 337-fold and 225-fold in EpiAirway™ and EpiDerm™, respectively. To determine induction of Phase I activity, a substrate cocktail was incubated with the models and metabolites were determined by LC/MS or fluorimetry. Baseline metabolism activity was very low. Significant induction of metabolic activity was observed only for the CYP1A1/CYP1B1 substrate. The results indicate that numerous changes in XME expression levels can be induced in epithelial tissues by nuclear receptor pathways. However, with the exception of CYP1A1/1B1, Phase I XME metabolic activity in human epidermal and airway epithelium appears to be low.

1-OH-bufuralol, 1-OH-midazolam, 4-OH-tolbutamide, 6-OH-clorzoxazone, ARE/EpRE activator, AhR activator, Biotransformation, CAR activator, CAR/PXR activator, CYP1A1, PPAR activator, Phase I XME Expression, Phase II XME Expression, Xenobiotic metabolizing enzyme (XME), qPCR

3-methylcholanthrene, Bufuralol, Chlorpromazine, Clofibric acid, Clorzoxazone, Midazolam, Rifampicin, T-buthydroquinone, Tolbutamide