The crucial role of particle surface reactivity in respirable quartz-induced reactive oxygen/nitrogen species formation and APE/Ref-1 induction in rat lung

Abstract

Persistent inflammation and associated excessive oxidative stress have been crucially implicated in quartz-induced pulmonary diseases, including fibrosis and cancer. We have investigated the significance of the particle surface reactivity of respirable quartz dust in relation to the in vivo generation of reactive oxygen and nitrogen species (ROS/RNS) and the associated induction of oxidative stress responses in the lung. Therefore, rats were intratracheally instilled with 2 mg quartz (DQ12) or quartz whose surface was modified by either polyvinylpyridine-N-oxide (PVNO) or aluminium lactate (AL). Seven days after instillation, the bronchoalveolar lavage fluid (BALF) was analysed for markers of inflammation (total/differential cell counts), levels of pulmonary oxidants (H2O2, nitrite), antioxidant status (trolox equivalent antioxidant capacity), as well as for markers of lung tissue damage, e.g. total protein, lactate dehydrogenase and alkaline phosphatase. Lung homogenates as well as sections were investigated regarding the induction of the oxidative DNA-lesion/oxidative stress marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) using HPLC/ECD analysis and immunohistochemistry, respectively. Homogenates and sections were also investigated for the expression of the bifunctional apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) by Western blotting and immunohistochemistry. Significantly increased levels of H2O2 and nitrite were observed in rats treated with non-coated quartz, when compared to rats that were treated with either saline or the surface-modified quartz preparations. In the BALF, there was a strong correlation between the number of macrophages and ROS, as well as total cells and RNS. Although enhanced oxidant generation in non-coated DQ12-treated rats was paralleled with an increased total antioxidant capacity in the BALF, these animals also showed significantly enhanced lung tissue damage. Remarkably however, elevated ROS levels were not associated with an increase in 8-OHdG, whereas the lung tissue expression of APE/Ref-1 protein was clearly up-regulated. The present data provide further in vivo evidence for the crucial role of particle surface properties in quartz dust-induced ROS/RNS generation by recruited inflammatory phagocytes. Our results also demonstrate that quartz dust can fail to show steady-state enhanced oxidative DNA damage in the respiratory tract, in conditions were it elicits a marked and persistent inflammation with associated generation of ROS/RNS, and indicate that this may relate to compensatory induction of APE/Ref-1 mediated base excision repair.

Figure 1

Myeloperoxidase activity in BALF of rat lungs 7 days following i.t. instillation of 2 mg DQ12 or DQ12 coated with either AL or PVNO. Data are expressed as mean ± SD (n = 5). *p < 0.01 vs. PBS (ANOVA, Tukey).

Figure 2

(A) H₂O₂ generation in the rat lung. H₂O₂levels were analysed spectrophotometrically in the BALF obtained from rats exposed to non-coated DQ12 or DQ12 coated with AL or PVNO (7 days after i.t. instillation). Data are expressed as mean ± SD (n = 5). *p < 0.01 vs. PBS (ANOVA, Tukey). (B) Correlation between H₂O₂ levels and total number of neutrophils in the BAL 7 days after i.t. instillation of 2 mg non-coated DQ12 or DQ12 coated with AL or PVNO.

Figure 3

Ex vivo release of H₂O₂ from PMA-stimulated BAL cells. BAL cells, obtained from rats exposed to non-coated DQ12 or DQ12 coated with PVNO or AL (7 days after i.t. instillation) were cultured in vitro (4 h) with or without PMA (100 ng/ml) to activate their oxidative burst. The graph shows the ratio between spontaneous and PMA-induced H₂O₂ production, which is expressed as % increase. Data are presented as mean ± SD (n = 3). *p < 0.01 vs. PBS.

Figure 4

Nitrite levels as detected in the BALF obtained from rats exposed to non-coated DQ12, or DQ12 coated with AL or PVNO (7 days after i.t. instillation). Data are expressed as mean ± SD (n = 5). *p < 0.05 vs. PBS.

Figure 5

Non-enzymatic total antioxidant capacity (TEAC) of BALF obtained from rats 7 days after i.t. instillation of non-coated DQ12 or DQ12 coated with AL or PVNO. Data are presented as mean ± SD (n = 5). *p < 0.01.

Figure 6

8-OHdG analysis by HPLC/ECD in lung tissue, obtained from rats exposed to 2 mg non-coated DQ12 or DQ12 coated with PVNO or AL (7 days after i.t. instillation). Data are presented as mean ± SD (n = 5). *p < 0.01 vs. PBS.

Figure 7

(A-D) Representative images of lung sections, obtained from controls (A) or rats exposed to 2 mg non-coated DQ12 (B) or DQ12 coated with PVNO (C) or AL (D), 7 days after i.t. instillation, stained with an antibody against 8-OHdG (original magnification × 400, original magnification of inserts × 1000). E: Positive cells were quantified in five random chosen areas (n = 4) at a magnification of × 1000.

Figure 8

Semi-quantitative analysis of APE/Ref-1 Western blots of lung homogenates of 5 animals per group exposed to PBS, 2 mg non-coated DQ12, or DQ12 coated with PVNO or AL 7 days after a single i.t. instillation. Data are presented as mean ± SD (n = 4). *p < 0.01 vs. PBS.

Figure 9

(A-D): Representative images of lung sections, obtained from a control rat (A) or rats exposed to 2 mg non-coated DQ12 (B) or DQ12 coated with PVNO (C) or AL (D), 7 days after i.t. instillation, stained with an antibody against APE/Ref-1 (original magnification × 400, original magnification of inserts × 1000. (E) Positive stained cells were quantified in five random areas of the left lung lobe of four animals per group at a magnification of × 1000.

Figure 10

(A) Representative image of Western blot investigation of the expression of APE/Ref-1 protein in nuclear and cytosolic extracts of alveolar macrophages (NR8383) as well as rat lung epithelial cells (RLE) cultured in complete medium. (B-C) Immunocytochemical image of (B) APE/Ref-1 staining and (C) Hoechst nuclear counter-staining in RLE cells. Original magnification of × 1000.