Human keratinocyte response to 4,4'-methylene diphenyl diisocyanate-glutathione conjugate exposure

Abstract

Workplace exposure to diisocyanates like 4,4'-methylene diphenyl diisocyanate can cause occupational asthma (MDI-OA), and the underlying biological pathways are still being researched.Although uncertainty remains, evidence supports the hypothesis that dermal exposure to MDI plays an important role in the development of MDI-OA.Gene expression, proteomics, and informatics tools were utilised to characterise changes in expression of RNA and protein in cultured human HEKa keratinocyte cells following exposure to conjugates of MDI with glutathione (MDI-GSH).RT-qPCR analysis using a panel of 39 candidate primers demonstrated 9 candidate genes upregulated and 30 unchanged.HPLC-MS/MS analysis of HEKa cell lysate identified 18 540 proteins across all samples 60 proteins demonstrate statistically significant differential expression in exposed cells, some of which suggest activation of immune and inflammatory pathways.The results support the hypothesis that dermal exposures have the potential to play an important role in the development of MDI-OA. Furthermore, proteomic and gene expression data suggest multiple immune (adaptive and innate) and inflammatory pathways may be involved in the development of MDI-OA.

Keywords: 4’-methylene diphenyl diisocyanate (MDI); human keratinocytes; isocyanates; label-free quantitative proteomics; occupational asthma (OA).

Figure 1.

In vitro MDI-GSH conjugate exposure upregulates specific cytokines, chemokines, alarmins, and transcription factors in 24 h exposed cultured HEKa cells. Total RNA was isolated from cultured HEKa cells from either non-exposed (GSH only) or MDI-GSH exposed cells by miRVana^™ miR isolation kit, reverse transcribed, and subjected to taqMan RT-qPCR. cytokines, chemokines, alarmins, and transcription factor mRna expression of (a) IL33, (b) TSLP, (c) GMCSF, (D) CCL22, (e) IL1B, (F) CXCL10, (G) PPARG, (H) MCPIP, and (i) NFATC2 were determined (N = 7; bars, s.e.m) (*p < 0.05, **p < 0.01, ***p < 0.001). total Rna isolated from HEKa cells collected 24 h post-exposure to MDI-GSH conjugates in complete culture media exposure.

Figure 2.

+ESI mass spectrum of the MDI-GSH conjugation reaction products.

Figure 3.. MS/MS analysis of m/z 532.1857 and predicted MS/MS fragments of MDI-GSH conjugate.

Four predicted fragments were identified in the MS/MS of m/z 532.1857 including [M + H]⁺ =179.0485, [M + H]⁺=199.1228, [M + H]⁺=225.1020, and [M + H]⁺=233.0588.