Even though the skin is the largest organ in the human body, its metabolic activity is often overlooked. The fact is, however, that xenobiotics can enter into systemic circulation through the epidermis and dermis either by direct absorption or metabolic activation. While enzymatic activity in the skin is much lower relative to the liver, especially for phase I CYP450s, many other phase I non-CYP and phase II enzymes are expressed, including flavin containing monooxygenases, alcohol and aldehyde dehydroxygenases, aldo-keto reductases, glutathione transferases, N-acetyl transferases and UDP glucuronosyltransferases. Activated keratinocytes have been reported to show increased expression of drug metabolising enzymes.
Evaluation of Phase I metabolism in the skin is especially important for drugs and chemicals designed for topical application because of the risk that protein reactive metabolites formed in the skin may contribute to or cause skin sensitisation. To ameliorate some of the obstacles, such as expense, associated with freshly excised human skin or other reconstructed 3D models, we chose to examine if human skin S9 or the human keratinocyte cell line presented a viable, low-cost alternative that would provide more relevant data than liver S9.
Nine substrates, including compounds known to cause skin sensitisation after bioactivation, as well as Phase I and Phase II markers, were evaluated in human skin S9, human liver S9 and HaCaT cells, which are a human keratinocyte cell line. The S9 was supplemented with the different cofactors, NADPH, UDPGA, acetyl CoA and GSH, for CYP-mediated metabolism, glucuronidation, acetylation and glutathione conjugation, respectively. The substrates were incubated with cofactors for up to 120 minutes in the S9 fractions and up to 24 hours in the HaCaT cells. Substrate depletion was monitored using LC-MS/MS and metabolite identification was performed using a QTof LC-MS/MS.
Unsurprisingly, liver S9 showed the highest levels of metabolic activity. HaCaT cells were more metabolically active for all substrates and enzymes when compared with the skin S9 with the exception of one substrate (phthalazine, a substrate for aldehyde oxidase). Differential metabolism was also observed when compared to liver S9. These results illustrate the potential application of the HaCaT human keratinocyte cell line in evaluating skin metabolism. This is relevant to the Cosmetic Europe Skin Tolerance Task Force testing strategy for skin sensitisation potency prediction, and should help in introducing bioavailability and metabolism into an integrated approach for skin safety assessment.