Waterpipe smoke and e-cigarette vapor differentially affect circadian molecular clock gene expression in mouse lungs

Abstract

The use of emerging tobacco products, such as waterpipe or hookah and electronic cigarettes (e-cigs), has gained significant popularity and are promoted as safer alternatives to conventional cigarettes. Circadian systems are internal biological oscillations that are considered important regulators of immune functions in mammals. Tobacco induced inflammatory lung diseases frequently exhibit time-of-day/night variation in lung function and symptom severity. We investigated the impact of inhaled e-cig vapor and waterpipe smoke (WPS) on pulmonary circadian molecular clock disruption by determining the changes in expression levels and abundance of core clock component genes (BMAL1, CLOCK) and clock-controlled output genes (Rev-erbα, Per2, Rev-erbβ, Cry2, Rorα) in mouse lungs. We showed that the expression levels of these pulmonary core clock genes and clock-controlled output genes were altered significantly following exposure to WPS (Bmal1, Clock, and Rev-erbα). We further showed a significant yet differential effect on expression levels of core clock and clock-controlled genes (Bmal1, Per2) in the lungs of mice exposed to e-cig vapor containing nicotine. Thus, acute exposure to WPS and e-cig vapor containing nicotine contributes to altered expression of circadian molecular clock genes in mouse lungs, which may have repercussions on lung cellular and biological functions.

Fig 1. Acute waterpipe smoke (WPS) exposure altered the levels of circadian molecular clock proteins in mouse lung.

C57BL/6J mice were exposed to air or WPS for 10 days and euthanized 24-hrs post-last exposure. Abundance of clock and clock-controlled proteins were analyzed in whole lung homogenate samples by immunoblot analysis. β-Actin bands shown for BMAL-1 and CLOCK are identical as both proteins were probed on the same membrane. The band intensity was measured by densitometry analysis and data were presented as fold change relative to β-actin loading control. Ten out of the twelve samples from the full gel/blot were included in the figure (S1 Fig). Full gels/blots with bands (unedited/uncropped electrophoretic gels/blots) derived from the same experiments were shown (S1 Fig). Data are shown as mean ± SEM, n = 5 per group. Statistical significance was calculated using the unpaired Student’s t-test *P < 0.05, **P < 0.01 vs. air controls.

Fig 2. Acute exposure to inhaled e-cig vapor containing nicotine showed altered levels of circadian molecular clock proteins in mouse lung.

C57BL/6J mice were exposed to e-cig vapor containing nicotine (25mg/ml) or propylene glycol (PG) alone for three days (2 hrs/day) and euthanized 2-hrs post-last exposure on day 3. Abundance of circadian clock proteins was measured in lung homogenates by immunoblot analysis. β-Actin bands shown for BMAL-1 and PER-2 are identical as both proteins were probed on the same membrane. Similarly, β-Actin bands shown for CLOCK and REV-ERBα are identical as both proteins were also probed on the same membrane. The band intensity was measured by densitometry analysis and data were presented as fold change relative to β-actin loading control. Full gels/blots with bands (unedited/uncropped electrophoretic gels/blots) derived from the same experiments were shown (S2 Fig). Data are shown as mean ± SEM, n = 4 for air and PG+Nicotine group and n = 5 for PG group. Only four of the five samples in the PG+Nicotine group from each full gel/blot targeted for each protein were included in the figure (S2 Fig). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. *P < 0.05, **P < 0.01 vs. air or PG.

Fig 3. ELISA analysis showed altered levels of circadian molecular clock proteins in mice exposed to acute WPS.

C57BL/6J mice were exposed to air or WPS for 10 days and euthanized 24 hrs post-last exposure. Levels of BMAL1, NR1D1 (REV-ERBα), and PER2 were measured in lung homogenates by ELISA as per manufacturer’s instructions. Data are shown as mean ± SEM, n = 4–7 per group except for BMAL1 ELISA we used n = 84 for air-exposed mice. Statistical significance was calculated using the unpaired Student’s t-test. **P < 0.01 vs. air controls.

Fig 4. ELISA analysis showed altered levels of circadian molecular clock proteins in mice exposed to inhaled e-cig vapor containing nicotine.

C57BL/6J mice were exposed to e-cig vapor containing nicotine (25 mg/ml), propylene glycol (PG) alone and air as a control (2 hrs/day) and euthanized 2 hrs post-last exposure on day 3. The levels of BMAL1, NR1D1 (REV-ERBα), and PER2, were measured in lung homogenates by ELISA as per manufacturer’s instructions. Data are shown as mean ± SEM, n = 5–6 per group.

Fig 5. Acute exposure to waterpipe smoke exhibited changes in the expression of circadian molecular clock genes.

C57BL/6J mice were exposed to air or WPS for 10 days and euthanized 24-hrs post-last exposure. mRNA expression of circadian clock genes was measured using qPCR analysis from RNA extracted from the lung tissues of mice exposed to WPS and air. Data are shown as mean ± SEM, n = 6–7 per group. Statistical significance was calculated using the unpaired Student’s t-test. *P < 0.05, **P < 0.01, vs. air controls.

Fig 6. Acute exposure to e-cig vapor containing nicotine alters the expression of circadian molecular clock genes.

C57BL/6J mice were exposed to e-cig vapor containing nicotine (25 mg/ml) or propylene glycol (PG) alone for 3 days (2 hrs/day) and euthanized 2 hrs post-last exposure on day 3. mRNA expression of clock and clock-controlled genes were measured using qPCR analysis from the lung tissues of mice exposed to PG with nicotine, PG alone, and air as a control. Data are shown as mean ± SEM, n = 5 per group. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05, **P < 0.01 vs. PG alone or air controls.

Fig 7. Schematic representation of waterpipe smoke (WPS) and e-cigarette vapor containing nicotine-induced alterations in the expression and abundance of circadian molecular clock genes.

Exposure to alternative tobacco products, such as (A) WPS and (B) e-cig vapors containing propylene glycol with nicotine affects the expression of circadian molecular clock genes in mouse lungs. Molecular clock consists of a heterodimer of transcription factors BMAL1and CLOCK. The BMAL1:CLOCK heterodimer starts transcription of Per2 and Cry2 by binding to specific DNA elements (E-box) located in the promoter region. Altered expression of circadian clock genes may be a result of acute WPS and/or e-cig exposure induced oxidative stress and lung inflammatory response. This can negatively affect pulmonary function during chronic exposures and thus may contribute to the pathobiology of chronic airway diseases. BMAL1, brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1; CLOCK, circadian locomotor output cycles protein kaput; nuclear receptor subfamily 1 group D member 1 (Nr1d1 or Rev-erbα); nuclear receptor subfamily 1, group D, member 2 (Nr1d2 or Rev-erbβ); Rorα, retinoic acid receptor-related orphan receptor α; period circadian regulator 2 (Per2); Cryptochrome 2 (Cry2).