TiO2 Nanoparticles synergistically increase LPS-induced NO Production by Macrophages

Although TiO2 NPs particles are endowed with little acute toxicity in vitro, they exert pro-inflammatory effects when inhaled in vivo. In this study, we evaluated the effects of several TiO2NPs, alone or in combination with LPS, on Raw264.7 macrophages in terms of cell viability (determined up to 72h with resazurin assay), NO production (estimated by nitrite concentration in the culture medium after 48h and 72h of treatment), and Nos2 induction, assessed with RT-PCR after 24h of treatment. We tested three types of TiO2 NP: a) pristine TiO2 NP of industrial origin (84% anatase/16% brookite, average NP size 45 nm), b) citrate-coated TiO2 NP, derived from pristine TiO2 NPs (average NP size 64 nm), c) Aeroxide P25 TiO2 NPs (80% anatase/20% rutile, average NP size 25 nm). All the TiO2 NPs did not affect significantly cell viability even at the highest doses tested (IC50>80 µg/cm2 at 24,48 and 72h). On the contrary, all the NP preparations, at the dose of 20 µg/cm2, induced Nos2. In particular, Nos2 was 3-fold, 8-fold and 4-fold induced by pristine, citrate coated and Aeroxide P25 TiO2 NPs alone, while fold-induction rose to 13 for pristine and citrate-coated NPs and to 30 for Aeroxide P25 NPs in the presence of 1 ng/ml LPS. LPS alone caused a 6-fold increase in Nos2 expression. The effects of pristine, citrate-coated and Aeroxide P25 TiO2 NPs on NO production were clearly dose- and time-dependent. At 80 g/cm2 of NP, nitrites were 5-fold, 9-fold and 7-fold higher than in the untreated control at 48h and 7-fold,10-fold and 8-fold higher than control at 72h. For all the TiO2NPs tested, the No-Observed-Effect-Level (NOEL) was 20 g/cm2 at 48h and 10 g/cm2 at 72h. After 48h-incubation with LPS (1 ng/ml) and TiO2 (80 µg/cm2), nitrite medium concentration was 31-fold, 30-fold and 25-fold higher than control with, respectively, pristine, citrate-coated and Aeroxide P25. LPS alone caused a 22-fold increase in medium nitrites. Several TiO2 NPs significantly induce Nos2 gene expression and increase NO production in a dose- and time-dependent manner in Raw264.7 cells. The enhancement of Nos2 induction by LPS suggests that the pro-inflammatory effect of TiO2 NPs in vivo may be exacerbated by concomitant infectious conditions and surface interactions with bacterial endotoxins. Supported by EU Grant NMP4-SL-2012-280716 (Sanowork Project)