Chronic Exposure to a Triple-Toxicant Mixture Upregulates AP-1, Nrf2, And OPN to Exacerbate Oxidative Stress in Vivo

To elucidate the mechanisms of oxidative stress induced by 16- and 32-week exposures to ozone, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and dibutyl phthalate (DBP) in B6C3F1 mice, we analyzed the activation of activator protein‑1 (AP‑1), nuclear factor erythroid-2-related factor 2 (Nrf2) and osteopontin (OPN) in lung and liver tissues using EMSA and Western blotting. In the lungs, AP-1 DNA-binding activity was significantly higher in the co-exposure groups compared to single-toxicant treatments, peaking in the triple-combination group (ozone+NNK+DBP). Similarly, hepatic AP-1 activation was observed across all treatment groups at both time points, with combined exposures eliciting a stronger response than individual treatments at 16 weeks. Furthermore, the protein expression of AP-1 subunits (c-Fos, c-Jun, JunB, and JunD), Nrf-2 and OPN protein were upregulated in both organs across all treated groups. These findings suggest that the synergistic interplay of these toxicants heavily exacerbates chronic oxidative stress.