title: Glutathione metabolism in the HaCaT cell line as a model for the detoxification of the model sensitisers 2,4-dinitrohalobenzenes in human skin.
abstract: Glutathione (GSH) is the most prominent antioxidant in cells and the co-factor of an important set of enzymes involved in the skin metabolic clearance system, glutathione S-transferases (GST). Here, we describe an LC-MS (liquid chromatography-mass spectroscopy) method to measure GSH and its disulfide form (GSSG) in HaCaT cells and a 3D Reconstructed Human Epidermis (RHE) model. In our assay, the basal level of GSH in both systems was in the low nmol/mg soluble protein range, while the level of GSSG was systematically below our limit of quantification (0.1 μM). We found that 2,4-dinitrohalobenzenes deplete the GSH present in HaCaT cells within the first hour of exposure, in a dose dependent manner. The level of GSH in HaCaT cells treated with a single non-toxic dose of 10 μM of dinitrohalobenzene was also shown to increase after two hours. While cells treated with 1-chloro-2,4-dinitrobenzene (DNCB) and 1-fluoro-2,4-dinitrobenzene (DNFB) repleted GSH to levels similar to untreated control cells within 24h, 1-bromo-2,4-dinitrobenzene (DNBB) seemed to prevent such a repletion and appeared to be the most toxic compound in all assays. A mathematical modelling of experimental results was performed to further rationalise the differences observed between test chemicals. For this purpose the biological phenomena observed were simplified into two sequential events: the initial depletion of the GSH stock after chemical treatment followed by the repletion of the GSH once the chemical was cleared. Activation of the nuclear factor E2-related factor 2 (Nrf2) pathway was observed with all compounds within two hours, and at concentrations less than 10 μM. These data show that GSH depletion and repletion occur rapidly in skin cells and emphasize the importance of conducting kinetic studies when performing in vitro experiments exploring skin sensitization.
authors.affiliation: Unilever, Safety and Environmental Assurance Centre, Colworth Science Park, Sharnbrook, Bedfordshire, MK44 1LQ, UK. Electronic address: Sandrine.jacquoilleot@unilever.com.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Jacquoilleot
authors.affiliation: Unilever, Safety and Environmental Assurance Centre, Colworth Science Park, Sharnbrook, Bedfordshire, MK44 1LQ, UK.
authors.forename: David
authors.initials: D
authors.lastname: Sheffield
authors.affiliation: MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Sherrington Building, Ashton Street, The University of Liverpool, Liverpool L69 3GE, UK.
authors.forename: Adedamola
authors.initials: A
authors.lastname: Olayanju
authors.affiliation: MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Sherrington Building, Ashton Street, The University of Liverpool, Liverpool L69 3GE, UK.
authors.forename: Rowena
authors.initials: R
authors.lastname: Sison-Young
authors.affiliation: MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Sherrington Building, Ashton Street, The University of Liverpool, Liverpool L69 3GE, UK.
authors.forename: Neil R
authors.initials: NR
authors.lastname: Kitteringham
authors.affiliation: MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Sherrington Building, Ashton Street, The University of Liverpool, Liverpool L69 3GE, UK.
authors.forename: Dean J
authors.initials: DJ
authors.lastname: Naisbitt
authors.affiliation: Unilever, Safety and Environmental Assurance Centre, Colworth Science Park, Sharnbrook, Bedfordshire, MK44 1LQ, UK.
authors.forename: Maja
authors.initials: M
authors.lastname: Aleksic
mesh_terms: D000975:Antioxidants / Q000378:metabolism; D002460:Cell Line; D002853:Chromatography, Liquid; D003198:Computer Simulation; D004136:Dinitrobenzenes / Q000633:toxicity; D004137:Dinitrochlorobenzene / Q000633:toxicity*; D004139:Dinitrofluorobenzene / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
chemical_list: D000975:Antioxidants; D004136:Dinitrobenzenes; D004137:Dinitrochlorobenzene; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; C011688:1-bromo-2,4-dinitrobenzene; D004139:Dinitrofluorobenzene; D005982:Glutathione Transferase; D005978:Glutathione
keywords: Glutathione conjugation; HaCaT cell line; RHE skin model; Skin metabolism