Skin sensitisation prediction using in vitro screening methods

Abstract

My PhD research project was designed to bring innovation into in vitro methods for identification of industrial chemicals, particularly epoxy resin systems (ERS), with skin sensitising potential as a means to minimise the use of animals for this purpose. In my research, the generalisability of two in vitro methods originally developed and validated for identification of small molecules to assess the skin sensitising potential of ERS was assessed. Specifically, my research focussed on (i) the human cell line activation test (h-CLAT) which mimics the characteristics of Langerhans cells during the maturation process following their activation by chemical sensitisers, and (ii) the direct peptide reactivity assay (DPRA), that assesses the initial interaction between potential chemical sensitisers with human skin proteins. The ERS data generated using these in vitro methods were compared with the skin sensitisation data for the same compounds generated using the widely accepted murine local lymph node assay (LLNA) in order to gain insight into the accuracy and reliability of the in vitro methods. For h-CLAT, I optimised the assay conditions for a 96-well format using 1.6x105 cells/well as well as anti-CD54-FITC and anti-CD86-PE. The relative fluorescent intensity (RFI) of CD54 and CD86 on THP-1 cells was determined using three-coloured flow cytometry. A chemical was regarded as being a positive sensitiser if the RFI of CD54 was >200% and/or that for CD86 was >150%. Five ERS tested in the h-CLAT assay, viz bisphenol A diglycidyl ether (DGEBA), trimethylolpropane triglycidyl ether (TMPTGE), poly(ethyleneglycol) diglycidylether (PEGGE) tetraphenylolethae glycidyl ether (THETGE), and poly[(phenyl glycidyl ether)-co-formaldehyde] (PPGE) gave negative results. These findings imply that the h-CLAT assay undertaken in standard format may be unsuitable for assessing skin sensitisation potential of ERS as the CD54 and CD86 were not induced. To address this issue, I investigated the possibility that ERS induced cytokine release in the h-CLAT assay may be a more sensitive marker of skin sensitisation than changes in expression levels of CD54 and CD86 on THP-1 cells. Encouragingly, concentrations of the cytokines, IL-6 and IL-8, in the cultured THP-1 cell supernatant quantified using a Meso ScaleTM Discovery human pro-inflammatory multiplex immunoassay, were markedly increased in DGEBA, TMPTGE, THETGE and PPGE. For the DPRA, chemicals with known sensitising capacity were incubated with three synthetic heptapeptides, Cor1-C420 (Ac-NKKCDLF), heptapeptides containing cysteine (Ac-RFAACAA) and lysine (Ac-RFAAKAA) in order to determine the optimal experimental conditions. The sensitising potential of the chemicals were correlated with depletion of each heptapeptide individually in a reaction mixture. The applicability of the DPRA to assess the skin sensitising potential ERS was investigated together with known positive and negative control compounds. The aforementioned heptapeptides were selected as they had been previously shown to have a high correlation with LLNA data when used to assess small molecules. My DPRA findings show that the optimal incubation temperature for incubation of all heptapeptides was 25°C. Importantly, my data also show that the apparent heptapeptides depletion level is affected by the tube materials used for the DPRA. Specifically, Cor1-C420 was stable in polypropylene tubes but failed to meet the assay acceptance criteria for days 1-3 when borosilicate glass tubes were used. As for cysteine, it was not stable on day 3 post-incubation when glass was used for the assay. Although lysine was stable in both polypropylene and glass tubes during the course of the DPRA, the apparent extent of lysine depleted by the chemical, ethyl acrylate, differed between polypropylene (24.7 ± 5.8%) and glass (47.3 ± 7.7%) vials. Another novel finding was instability of the peptide-chemical complex (i.e. Cor1-C420-cinnamaldehyde and cysteine-2,4-dinitrochlorobenzene) suggesting that the complex formation may be partially reversible. This information suggests that data generated by the DPRA in high-throughput format involving the screening of hundreds of chemicals simultaneously, may not be accurate. Poor aqueous solubility of ERS in in vitro assays is a considerable challenge. To address this issue, the solubility of five ERS using a range of solvent:reaction buffer combinations was compared. In brief, a solvent comprising a 1:1 methanol:acetonitrile containing 1% tert-butanol was effective in solubilising these five ERS in reaction buffer. Using this optimised solvent system for dissolution of DGEBA, TMPTGE, THETGE and PPGE, the DPRA data generated were significantly correlated with the LLNA data on skin sensitisation with the exception that PEGGE was positive in the DPRA but classified as a non-sensitiser in the LLNA. In summary, my findings show that there are many challenges to be overcome in future work beyond the scope of my PhD research project in terms of adapting the DPRA and h-CLAT assays to high-throughput format in order to provide accurate information on the skin sensitisation potential of novel industrial chemicals.