Application of small molecule inhibitors to probe the mechanism underlying nanomaterial pulmonary inflammatory responses <i>in vitro</i>

Evidence shows that nanomaterial (NM)-induced pro-inflammatory cytokine production is regulated via redox-sensitive transcription factors (TFs). However, there is limited information regarding the intracellular signaling pathways that regulate NM-mediated activation of TFs. Gaining an understanding of the intracellular signaling pathways that are activated by NMs is key in developing our understanding of how NMs activate inflammatory responses. Traditionally used methods (e.g. immunostaining and western blotting) for assessing TF activation following NM exposure are often subjective and not amenable to high throughput screening. This paper proposes the application of the under-utilized method of selective small molecule inhibitors (SMIs) to rapidly probe the signaling pathways underpinning NM-induced inflammatory responses <i>in vitro</i>. The nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways were targeted for study as there is evidence that they are involved in NM-induced inflammation. In our study we demonstrate that silver (Ag), copper oxide (CuO) and zinc oxide (ZnO) NMs stimulate pro-inflammatory cytokine production by Calu-3 (epithelial) and J774A.1 (macrophage) cells <i>in vitro</i>. Inhibition of Inhibitor of NF-κB Kinase (IKK) reduced the pro-inflammatory response induced by Ag and ZnO NMs but had no significant effect on CuO NM-induced cytokine production. This data indicates that whilst Ag, ZnO and CuO NMs can all stimulate cytokine production by pulmonary cells <i>in vitro</i> they activate inflammatory responses via different mechanisms. We suggest that the wider adoption of SMIs can be used to provide a greater understanding of the mechanisms underlying NM toxicity to help identify what approaches can be used to screen NM toxicity.