Aldehyde Trapping by ADX-102 Is Protective against Cigarette Smoke and Alcohol Mediated Lung Cell Injury

Abstract

Most individuals diagnosed with alcohol use disorders smoke cigarettes. Large concentrations of malondialdehyde and acetaldehyde are found in lungs co-exposed to cigarette smoke and alcohol. Aldehydes directly injure lungs and form aldehyde protein adducts, impacting epithelial functions. Recently, 2-(3-Amino-6-chloroquinolin-2-yl)propan-2-ol (ADX-102) was developed as an aldehyde-trapping drug. We hypothesized that aldehyde-trapping compounds are protective against lung injury derived from cigarette smoke and alcohol co-exposure. To test this hypothesis, we pretreated mouse ciliated tracheal epithelial cells with 0-100 µM of ADX-102 followed by co-exposure to 5% cigarette smoke extract and 50 mM of ethanol. Pretreatment with ADX-102 dose-dependently protected against smoke and alcohol induced cilia-slowing, decreases in bronchial epithelial cell wound repair, decreases in epithelial monolayer resistance, and the formation of MAA adducts. ADX-102 concentrations up to 100 µM showed no cellular toxicity. As protein kinase C (PKC) activation is a known mechanism for slowing cilia and wound repair, we examined the effects of ADX-102 on smoke and alcohol induced PKC epsilon activity. ADX-102 prevented early (3 h) activation and late (24 h) autodownregulation of PKC epsilon in response to smoke and alcohol. These data suggest that reactive aldehydes generated from cigarette smoke and alcohol metabolism may be potential targets for therapeutic intervention to reduce lung injury.

Keywords: AUD; acetaldehyde; alcohol; cigarette; lung; malondialdehyde.

Figure 1

ADX-102 and bronchial epithelial cell viability. Medium release of lactate dehydrogenase (LDH) was measured and expressed as % viability in BEAS-2B cells (A) or LDH activity in 16HBE cells (B) after 24 h treatment with 0.1 µM to 10 mM ADX-102 in M-199 with 10% serum (Media). An equal number of cells were sonicated for maximum LDH release (Lysed). Positive assay control (+) was provided by a manufacturer. * p < 0.05 and ** p < 0.01 vs. 10 µM ADX-102; **** p < 0.0001 vs. 0–100 µM ADX-102. Bars represent SEM of biological n of three with three technical replicates. ns = not significant.

Figure 2

ADX-102 and bronchial epithelial cell migration. Circular wounds in monolayers of BEAS-2B cells were measured for cell migration into the wound area in the presence of 0–100 µM ADX-102 over time (A). Positive control for wound closure was M-199 with 10% serum (FBS). In panel (B), BEAS-2B cells were wounded in the presence of 5% cigarette smoke extract (CSE) and in the presence of 0–100 µM ADX-102, and % wound closure was measured. * p < 0.05 vs. 0 µM ADX-102. Bars represent SEM of n = 5, each with three replicates.

Figure 3

ADX-102 and bronchial epithelial cell protein kinase C alpha (PKCα) activity. BEAS-2B cells were pretreated with or without 10 µM ADX-102 for 1 h prior to treatment with M-199 containing 10% serum (Media) or 5% cigarette smoke extract (CSE) for 1 h. In the absence of ADX-102, **** p < 0.0001 CSE vs. Media. In the presence of CSE, ** p < 0.01. No ADX vs. ADX. Bars represent SEM of n = 9, each with three replicates.

Figure 4

ADX-102 and tracheal epithelial ciliated cell protein kinase C epsilon mediated cilia beating. Ciliated MTECs were treated with or without the combination of 5% cigarette smoke extract (CSE) and 50 mM ethanol (EtOH) in the presence or absence of 10 µM ADX-102, and cilia beat frequency (CBF) was measured from 0–24 h (A). Baseline control consisted of DMEM with 10% serum (Media). * p < 0.05 ADX vs. no ADX in the presence of CSE + EtOH at 3, 6, and 24 h. Dose–response (0–100 µM) for ADX-102 on combined CSE and EtOH stimulated (3 h) and autodownregulated (24 h) protein kinase C epsilon (PKCε). * p < 0.01 vs. medium control. Bars represent SEM of n = 6, each with three replicates (B).

Figure 5

ADX-102 and loss of tracheal epithelial cell cilia. Ciliated MTECs were treated with a combination of 5% cigarette smoke extract (CSE) and 50 mM ethanol (EtOH) in the presence or absence of 10 µM ADX-102, and the average number of motile cilia were measured at 3 (A) and 24 h (B). **** p < 0.01 vs. medium control or presence of ADX. Bars represent SEM of at least n = 9 individual experiments. ns = not significant.

Figure 6

ADX-102 and bronchial epithelial cell permeability. 16HBE cells were grown to confluence until maximal barrier function (Resistance) achieved. Cells were treated with either DMEM and 10% serum (Media), 5% cigarette smoke extract (CSE), 50 mM alcohol (EtOH), or the combination of smoke and alcohol for up to 72 h in the absence (A) or presence (B) of 10 µM ADX-102. * p < 0.02 vs. media at 72 h. Bars represent SEM of n = 5, each with three replicates.

Figure 7

ADX-102 and MAA adduct formation. 16HBE cells were grown to confluence in 60 mm culture dishes. Cells were treated with either M-199+10% serum (Media) or a combination of 5% cigarette smoke extract (CSE) and 50 mM alcohol (EtOH) for 72 h in the absence or presence of 10 µM ADX-102. * p <0.04 and ** p < 0.003 at 72 h. Bars represent SEM of n = 6.

Figure 8

Model diagram for ADX-102 aldehyde-trapping action on cigarette smoke and alcohol mediated injury to airway epithelial cell function.