Evaluating Isocyanate Toxicity using Skin Cells

Isocyanates are a family of highly reactive, low molecular weight chemicals widely used to produce polyurethane products such as adhesives, spray paints, insulation, resins, sealants, foams, and surface coatings. Currently, in the US, an estimated 440,000 workers are exposed to isocyanates via airborne aerosols, particles, or direct contact with the skin. Occupational isocyanate exposure can cause skin irritation, allergic contact dermatitis, as well as respiratory sensitization, and asthma.

In vitro cell-based toxicity models are valuable research tools to assess a substance’s toxicity. The ability of these models to reliably predict drug toxicity in humans is dependent on several factors, including the cell culture medium, which directly influences the growth and behavior of the target cells.

The authors of the publication we review here evaluate the toxicity profiles of two isocyanate precursors (HDI monomer and HDI isocyanurate) in the context of several different cell culture media formulations using cultured normal human skin cells. This is because these molecules have binding affinity to amino acids that are present in cell culture media and can potentially skew toxicity results.

The Lifeline® catalog offers a wide range of high-quality cell culture media kits optimized to culture many different cell types including the HBTECs used in the study reviewed here. Our media products do not contain antimicrobials and are phenol red-free to mitigate any undesirable, off-target effects they may cause to ensure the accuracy of your results.

• Human blood cell medium: RPMI Media
• Human endothelial cell media
  • VascuLife® VEGF Endothelial Cell Media and Vasculife EnGS Endothelial Cell Media
  • VascuLife VEGF-Mv Microvascular Cell Media and VascuLife EnGS-Mv Microvascular Cell Media
• Human epithelial cell media
  • Air-Liquid interface epithelial differentiation medium: HBTEC Media
  • Airway epithelial medium: BronchiaLife™ Media
  • Bladder epithelial medium: UroLife™ Media
  • Corneal epithelial medium: OcuLife™ Media
  • Female Reproductive medium: ReproLife™ Media and ReproLife CX Media
  • Keratinocyte medium: DermaLife K Media
  • Mammary epithelial medium: MammaryLife™ Media
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  • Renal epithelial medium: RenaLife™ Media
• Human fibroblast cell medium:
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• Human melanocyte medium: DermaLife M Media and DermaLife Ma Media
• Human neural stem cell medium: StemLife™ NSC Media
• Human smooth muscle cell medium: VascuLife SMC Media
• Human skeletal muscle cell medium: StemLife Sk Media
• Human stem cell media
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  • AdipoLife™ Dfkt-1 Adipogenesis Media and AdipoLife Dfkt-2 Adipogenesis Media
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New Research using Lifeline’s DermaLife Cell Culture Media

In a recently published study by Boyer and Colleagues, in vitro skin models were used to characterize the cytotoxicity of two isocyanates (HDI monomer and one of its trimers, HDI isocyanurate) that are components of clearcoat polyurethane paints. Isolated keratinocytes and melanocytes were cultured in Lifeline’s DermaLife Basal Media supplemented with LifeFactors DermaLife K or M, respectively. Neonatal foreskin fibroblasts were also isolated and cultured in DMEM/10% calf serum.

Three different media types were used to test the effect of amino acids and proteins in the media on the isocyanates toxicity due to their reactivity to amino groups: 1) cell-specific complete media (i.e., Dermalife Media K, M or DMEM/10% calf serum), 2) cell-specific basal media only (DermaLife Basal Media or DMEM) or 3) Hank’s balanced saline solution (HBSS Ca²⁺/Mg²⁺), which contains no proteins. The three media types for exposure contain different levels of amino acids and proteins ranging from 1.6 mg/mL to 7 mg/mL protein. Isocyanate stock solutions were prepared by diluting HDI monomer and HDI isocyanurate into DMSO to concentrations of 6 M and 0.1 M, respectively. The isocyanate DMSO solutions were diluted into test culture media (1:2000) prior to incubation with the cells.

The keratinocytes, fibroblasts, and melanocytes were incubated with different concentrations of the isocyanates for 18 hrs prior to cell viability quantification using the CellTiter-Glo® 2.0 ATP luciferase-based luminescence assay. This data was used to calculate the LC50 for each substance (concentration at which 50% of the cells die) and generate dose-response curves. Orthogonal cell viability and cell death assays, ApoTox-Glo^TM and CellTox Green^TM were also used.

Overall, a higher in vitro toxicity of cultured skin cells was observed with HDI isocyanurate trimer compared to HDI monomer exposure. Additionally, the cytotoxicity of the molecules was affected by the media compositions, where a decreased toxicity is observed in protein-rich media (increased LD50 values). Notably, HDI monomer toxicity was affected more by the presence of amino acids and protein in the media than HDI isocyanurate. The physiochemical difference in reactivity of the two forms of HDI to biological molecules most likely explains the observed toxicity differences and may have implications for skin penetration, adverse effects like skin sensitization, and systemic responses like asthma. Keratinocytes were found to be the most susceptible cell type to HDI isocyanurate toxicity while melanocytes were the most sensitive cell type to HDI monomer toxicity.

Because HDI monomer toxicity was more affected by the presence of protein in the culture media than HDI isocyanurate toxicity, experiments conducted with protein-free media would result in a significant under-estimation of their relative toxicities. Conclusions based on comparative toxicity studies and consequently inferences made to predict human toxicity would not be accurate. This publication highlights the importance of conduct toxicity testing in multiple media types particularly in the evaluation of compounds that may react with media components.

Have you used Lifeline’s cell culture media for your research program? If so, contact us and your study could be featured on our blog!