Strain- and sex-dependent pulmonary toxicity of waterpipe smoke in mouse

Abstract

Waterpipe smoking is emerging as a form of tobacco smoking, but its lung health/risks is not known. It has been shown that different mouse strains show differences in susceptibility to tobacco smoke. However, the effect of waterpipe smoke (WPS) exposure and strain differences in susceptibility to oxidative and inflammatory responses is not known. Here, we showed acute WPS exposure induced oxidative stress and inflammatory response in C57BL/6J and BALB/cJ mouse strains. WPS exposure induced inflammatory cell influx (neutrophils and T-lymphocytes) in bronchoalveolar lavage fluid (BAL fluid), which varied among mouse strains. Proinflammatory cytokines release differed among both the strains, but was significantly increased in C57BL/6J mice. Myeloperoxidase levels in BAL fluid were increased significantly in both the strains. Total reduced glutathione (GSH) level was decreased, whereas the level of oxidized or glutathione disulfide (GSSG) increased in lungs of both the strains. Similarly, the level of lipid peroxidation markers, 15-isoprostane (plasma), malondialdehyde and 4-hydroxy-2-nonenal (lung homogenates) were increased by WPS. Our data suggest that, oxidative stress and inflammatory responses are influenced by strain characteristics during acute WPS exposure. Overall, C57BL/6J mice showed more susceptibility to oxidative stress and inflammatory responses compared to BALB/cJ mice. Acute WPS mediated pulmonary toxicity is differentially regulated in different mouse strains.

Keywords: Inflammation; glutathione; lipid peroxidation; oxidative stress; waterpipe (hookah).

Figure 1

Schematic of laboratory waterpipe/hookah exposure setup for in vivo mouse exposure model. The waterpipe/hookah smoking system is setup for mouse exposure, which contains two waterpipe smoking assembly connected to the FMI pump that controls the timed flow of WPS along with dilution air into the exposure chamber using a computer controlled program. The pump is setup to a defined puff topography (e.g., ~171 puffs/h, each puff is about 3−4 sec. long with 17 sec. puff interval) as reported previously (Hakim et al. 2011). The flow rate of the pump which draws WPS was setup to 1.0 L/min with a constant flow of dilution air (filtered air) into the chamber (at the rate of 0.6 L/min). The exposure chambers were made of plexiglass, containing wired cages with cubical for individual mice. The arrows in the schematic indicates directionality of WPS and dilution air flow into the exposure chamber. WPS, Waterpipe Smoke

Figure 2

Acute WPS exposure induces pulmonary inflammation characterized by increased neutrophils and T‐lymphocytes in the BAL fluid. The inflammatory influx of (A) total cell counts, (B) macrophages, (C, E, and G) neutrophils, and (D, F, and H) lymphocytes into the BAL fluid of different strains of mice in response to 10 days WPS exposure. Total cells counts/mL were determined in BAL fluid by AO/PI staining using a cellometer. A total of 500 cells were counted to determine the total number of macrophages, neutrophils and lymphocytes on slides stained with Diff‐Quik. Data are represented as means ± SEM (n = 8 per group) and significance determined using two‐way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001 versus air.

Figure 3

Acute WPS exposure increased proinflammatory cytokines release in the BAL fluid. C57BL/6J and BALB/cJ mice were exposed to air or WPS for 10 days and euthanized 24 h post last exposure. (A–L) Proinflammatory mediators from air or WPS exposed mice were measured in the BAL fluid by Luminex multiplex assay (Bio‐Rad). Data are shown as mean ± SEM (n = 8 per group) and significance determined using two‐way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, versus air. ^{# #} P < 0.01; ^{# # #} P < 0.001 versus WPS.WPS, Waterpipe Smoke

Figure 4

Effect of acute WPS exposure on proinflammatory mediators in male and female C57BL/6J and Balb/cJ mice. Male and female mice of (A–E) C57BL/6J and (F–G) BALB/cJ strains were exposed to air or WPS for 10 days and euthanized 24 h post last exposure. Proinflammatory mediators from air or WPS exposed mice were measured in the BAL fluid by Luminex multiplex assay (Bio‐Rad). Data are shown as mean ± SEM. (n = 4 per group) and significance determined using two way ANOVA. Significant statistical differences between the groups are indicated by symbols above the bars as: *Sex difference within the strain.*P < 0.05, **P < 0.01 versus air. WPS, Waterpipe Smoke

Figure 5

Acute WPS exposure alters oxidative stress markers in mouse Lung. C57BL/6J and BALB/cJ mice were exposed to air or WPS for 10 days and euthanized 24 h. post‐last exposure. (A) MPO in BAL fluid, (B) Reduced glutathione and (C) Oxidized Glutathione in lung homogenate, (D) 15‐isoprostane levels in plasma, (E) : 4‐Hydroxy‐2‐nonenal and (F) Malondialdehyde in lung homogenate were measured by ELISA according to manufacturer's instructions. Data are shown as mean ± SEM (n = 8–12 per group) and significance determined using two‐way ANOVA. *P < 0.05 versus air. #P < 0.05 versus WPS.WPS, Waterpipe Smoke.