Identification of Cytochrome P450 2S1 in HaCaT Keratinocytes as a Responce to Surfactant Exposure

Description

Taking the cell proteomic response to different surfactants exposure as a base of our study, we assessed the eligibility of using human HaCaT keratinocytes culture as a test-system for studying the cytotoxicity of xenobiotics. As a models of toxicants, anionic surfactant and non-ionic surfactant were chosen. Overall, 21 proteins associated with cell detoxification and xenobiotics metabolism were detected, including orphan Cytochrome P450 2S1 (CYP2S1). According to the literature data, CYP2S1 induction in HaCaT was registered only by an increase in mRNA levels. The NextProt human proteome project database also did not contain information on the presence of CYP2S1 in keratinocytes at the protein level. The acquired data on quantitative and qualitative proteomics landscape change of HaCaT culture may be used for the revealing specific proteins/metabolic pathways related to the processes of metabolism of a wide spectrum of xenobiotics. Thus, the presented results draw a new prospect of using the HaCaT keratinocytes as a model of human epidermis for studying the metabolism of drugs/toxicants in human skin in vitro.

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HaCaT cells (from Bank of DKFZ, Heidelberg) were seeded in T75 tissue culture flasks and cultured in DMEM supplemented with 10% FBS and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin). Cells were kept at 37 °C in a humidified 5% CO2 atmosphere. The medium was changed every other day. After growing to ~60–70% confluence, the cultured cells were divided into four groups: control (fresh medium was added), anionic surfactant, and non-ionic surfactant in two concentrations. The medium was aspirated 48 hours later, and the cells were washed with PBS, trypsinized with 3 mL of 0.25% trypsin-EDTA per flask, and incubated briefly at 37 °C. The cells were microscopically examined to ensure they were completely detached before being transferred to a centrifuge tube. The cells were centrifuged at 1200 rpm for 5 min before being washed three times in ice-cold PBS. Finally, PBS was aspirated, and the pellet was resuspended in ice-cold deionized water. The water cell homogenate of HaCaT keratinocytes (180 μL of cold water, 65 mM DDT, and 1% protease inhibitor E64, freshly prepared every time) was prepared by sonication. The lysate was centrifuged at 15,000 × g at + 4 °C for 15 min twice to remove debris. The pair of water extracts (175 μg of protein) for each study group were in-solution digested with trypsin. Separation and identification of the peptides were performed on an Ultimate 3000 nano-flow HPLC system (Dionex, USA), connected to a Orbitrap Q Exactive mass-spectrometer (Thermo Scientific, USA) equipped with a Nanospray Flex NG ion source (Thermo Scientific, USA).

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