Glucose 6 phosphate (g6p)

The 3R4F cigarette was kindly provided by the Korea Institute of Toxicology. The TL and TW were purchased from Korean commercial sources. Dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), 2-aminoanthracene, benzo[a]pyrene and cytochalasin B were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aroclor 1254-induced Sprague Dawley rat liver S9 was obtained from Moltox (Boone, NC, USA). The S9-cofactor, consisting of phosphate buffer, NADP, glucose 6-phosphate, KCl, MgCl₂ and CaCl₂, was purchased from Wako (Tokyo, Japan).

For the analysis of G6Pase activity, the harvested livers were homogenised in ice-cold saline and these homogenates were centrifuged (600 g, 10 min, at 4 °C). The supernatants were collected, aliquoted, and stored at −80 °C until analysis. The liver lysates were incubated for 15 min at 37 °C in 65 mM sodium cacodylate (Wako Pure Chemical Industries, Osaka, Japan) buffer (pH 6.5) containing 10 mM EDTA and 10 mM glucose-6-phosphate (Wako Pure Chemical Industries, Osaka, Japan). The reaction was stopped by adding ice-cold 10% trichloroacetic acid (Wako Pure Chemical Industries, Osaka, Japan) and the samples were centrifuged (600 g, 10 min, at 4 °C). The supernatants were incubated for 10 min at 37 °C with ammonium molybdate (Wako Pure Chemical Industries, Osaka, Japan) and 1-amino-2-naphthol-4-sulfonic acid (Wako Pure Chemical Industries, Osaka, Japan). After adding triethanolamine (Wako Pure Chemical Industries, Osaka, Japan) solution, the absorbance was measured at 780 nm.

To analyze NO released from In₂O₃-treated cells, we measured the concentration of its products, nitrate (NO₃^-) and nitrite (NO₂^-), in the culture supernatant by using the Griess method. RAW 264.7 cells (5 × 10⁵ cells/ml) were seeded in 1.2 ml of phenol red-free DMEM (Gibco/BRL) containing 5% (v/v) FBS and 100 mg/l kanamycin in 6 Well Clear Multiwell Plates (BD Falcon). Then the cells were treated with 20 μg/mlIn₂O₃ for 2 or 4 h at 37°C. The culture supernatant was collected and centrifuged at 40,000 × g for 10 min at 4°C to remove In₂O₃ particles. To reduce NO₃^- to NO₂^-, the supernatant was incubated with 0.1 units/ml of nitrate reductase from Aspergillus niger (Sigma-Aldrich) in the presence of 1 mM glucose-6-phosphate (Wako Pure Chemical Industries, Osaka, Japan), 0.3 units/ml of glucose-6-phosphate dehydrogenase and 20 μM NADPH (Oriental Yeast, Tokyo, Japan) for 30 min at room temperature. The reaction mixture was incubated with 0.25% (w/v) sulfanilamide (Griess reagent I, Wako) and 0.025% (w/v) naphthylethylenediamine (Griess reagent II, Sigma-Aldrich) in 0.625% (v/v) phosphoric acid for 10 min at room temperature. The absorbance was measured at 540 nm with a microplate reader (Model 680, Bio-Rad laboratories) and NO₂^- concentration was determined by comparison with a standard curve generated with sodium nitrate (NaNO₂, Wako).