Oil Sands Process Water and Naphthenic Acids Activate Mammalian Macrophages via a Toll-Like Receptor 4 (TLR4)-Dependent Mechanism

Oil sands process water (OSPW), a byproduct of bitumen extraction, is a complex mixture of organic and inorganic compounds, including naphthenic acids (NAs), which have been linked to toxicity. In this study, a mixture of commercial NAs (cNAs) was used as a surrogate for OSPW-derived NAs to examine their immunotoxic effects. Exposure of macrophage cell lines to OSPW and cNAs significantly induced secretion of proinflammatory cytokines, including interleukin (IL)-1β, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1. Pharmacological inhibition of Toll-like receptor 4 (TLR4) and its coreceptor MD2 markedly reduced these responses, indicating a TLR4/MD2-dependent mechanism. Human embryonic kidney (HEK293) cells stably expressing the human TLR4 complex responded to OSPW and cNAs by secreting elevated levels of IL-8, a response that was also inhibited by TLR4 blockers. Finally, Jurkat TLR reporter cells confirmed that cNA exposures activate NF-κB signaling specifically via TLR4 (NF-κB:eGFP:TLR4), as no response was observed in cells expressing the TLR5 or TLR2/6 heterodimer. These results provide mechanistic evidence for TLR4-dependent sensing of NAs and highlight the utility of immune cell-based bioassays in evaluating the immunotoxic potential of organic pollutants in complex environmental samples.