A food-born heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), suppresses tumor necrosis factor-α expression in lipoteichoic acid-stimulated RAW 264.7 cells

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine with strong carcinogenic and mutagenic potential, is created abundantly in the overcooking of meat and fish. Carcinogenic toxicants are often implicated in immunosuppression, where cancer cells are not easily eliminated by the host immune system. Here, we investigated the effect of PhIP on tumor necrosis factor-alpha (TNF-α) expression by murine macrophage-like cells (RAW 264.7) stimulated with lipoteichoic acid (LTA), a major virulence factor of Gram-positive bacteria. Upon exposure to LTA purified from Staphylococcus aureus, TNF-α expression was substantially induced, whereas pretreatment with PhIP significantly inhibited LTA-induced TNF-α expression. LTA is known to activate Toll-like receptor 2 (TLR2) and NF-κB, resulting in TNF-α expression. Interestingly, PhIP did not interfere with LTA-binding to TLR2, its stimulation of TLR2, or the DNA-binding activity of NF-κB. However, treatment with actinomycin D facilitated the PhIP-induced attenuation of TNF-α mRNA expression, implying that PhIP might decrease TNF-α mRNA stability rather than its biosynthesis. Furthermore, Western blot analysis demonstrated that PhIP reduced the phosphorylation of ERK1/2 and JNK but not p38 kinase in LTA-stimulated cells. The addition of a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, rescued PhIP-inhibited TNF-α expression in LTA-stimulated cells. These results suggest that PhIP downregulates TNF-α expression in LTA-stimulated macrophages by decreasing TNF-α mRNA stability and signaling pathways related to PKC, ERK1/2, and JNK activation.