Elsevier

Alcohol

Volume 46, Issue 1, February 2012, Pages 51-59
Alcohol

Malondialdehyde–acetaldehyde-adducted protein inhalation causes lung injury

https://doi.org/10.1016/j.alcohol.2011.09.001Get rights and content

Abstract

In addition to cigarette smoking, alcohol exposure is also associated with increased lung infections and decreased mucociliary clearance. However, little research has been conducted on the combination effects of alcohol and cigarette smoke on lungs. Previously, we have demonstrated in a mouse model that the combination of cigarette smoke and alcohol exposure results in the formation of a very stable hybrid malondialdehyde–acetaldehyde (MAA)-adducted protein in the lung. In in vitro studies, MAA-adducted protein stimulates bronchial epithelial cell interleukin-8 (IL-8) via the activation of protein kinase C epsilon (PKCɛ). We hypothesized that direct MAA-adducted protein exposure in the lungs would mimic such a combination of smoke and alcohol exposure leading to airway inflammation. To test this hypothesis, C57BL/6J female mice were intranasally instilled with either saline, 30 μL of 50 μg/mL bovine serum albumin (BSA)–MAA, or unadducted BSA for up to 3 weeks. Likewise, human lung surfactant proteins A and D (SPA and SPD) were purified from human pulmonary proteinosis lung lavage fluid and successfully MAA-adducted in vitro. Similar to BSA–MAA, SPD–MAA was instilled into mouse lungs. Lungs were necropsied and assayed for histopathology, PKCɛ activation, and lung lavage chemokines. In control mice instilled with saline, normal lungs had few inflammatory cells. No significant effects were observed in unadducted BSA- or SPD-instilled mice. However, when mice were instilled with BSA–MAA or SPD–MAA for 3 weeks, a significant peribronchiolar localization of inflammatory cells was observed. Both BSA–MAA and SPD–MAA stimulated increased lung lavage neutrophils and caused a significant elevation in the chemokine, keratinocyte chemokine, which is a functional homologue to human IL-8. Likewise, MAA-adducted protein stimulated the activation of airway and lung slice PKCɛ. These data support that the MAA-adducted protein induces a proinflammatory response in the lungs and that the lung surfactant protein is a biologically relevant target for malondialdehyde and acetaldehyde adduction. These data further implicate MAA-adduct formation as a potential mechanism for smoke- and alcohol-induced lung injury.

Introduction

Heavy alcohol use is associated with the increased occurrence of lung diseases, including pneumonia. In fact, meta-analyses have demonstrated that alcohol has a dose-dependent association as a risk factor for pneumonia (Samokhvalov et al., 2010). Part of the adverse health effect of alcohol emanates from direct exposure of the lungs to alcohol. Alcohol diffuses from the bronchial circulation into the airways where it is exposed to air, vaporizes, and then condenses back on to the airways. This “rain” effect results in both acute and chronic alterations to innate lung defense and mucociliary clearance (Sisson, 2007). Unfortunately, the vast majority (between 80 and 95%) of alcoholics also smoke cigarettes (Patten et al., 1996).

There are approximately 46.6 million adult cigarette smokers in the United States representing 20.6% of the US population with the Midwest population reaching as high as 23.1% (Centers for Disease Control and Prevention, 2010). Unfortunately, there has been no decline in this number from 2005 to 2009. One-third of heavy smokers are alcoholics (Miller and Gold, 1998). Although the association of cigarette smoking and alcohol consumption is universally recognized, relatively few coexposure studies exist compared with mechanistic studies of the biologic effects of either alcohol alone or smoke alone exposure on lung injury.

One shared mechanism for the pathogenesis of lung injury from alcohol and cigarette smoke is via reactive aldehyde formation. Alcohol is rapidly metabolized into acetaldehyde (AA) by the action of alcohol dehydrogenase (ADH). In addition to the AA released during the first pass from the liver into the bloodstream, both ADH and CYP2E1 are present in lung and act to metabolize alcohol. In addition, oxidative stress-induced lipid peroxidation occurs in the lung resulting in the alcohol-induced formation of malondialdehyde (MDA). Although lipid peroxidation-derived MDA formation can also be stimulated by cigarette smoke, the direct pyrrolysis of tobacco generates large concentrations of AA. These aldehydes are highly reactive and nonenzymatically bind to proteins covalently to form Schiff bases. Such aldehyde-protein binding in vivo generally results in a deleterious outcome because of the modification of nucleophilic residue side chains found on amino acids such as lysine and hydroxylysine (Harding, 1985). These reactions between aldehyde and proteins are commonly known as protein adducts.

The adduction of AA or MDA individually to proteins results in an unstable protein adduct with a short half-life. However, the formation of a hybrid protein adduct consisting of 2 moles of MDA and 1 mole of AA results in a very stable protein adduct known as the malondialdehyde–acetaldehyde protein adduct (MAA adduct; Fig. 1). Because cigarette smoke contains such high concentrations of AA and alcohol consumption leads to elevated MDA levels in the lung, the lungs of a smoker who drinks alcohol are the ideal milieu for the formation of MAA adducts.

Previously, we have demonstrated in a mouse model of combined cigarette smoke and alcohol exposure that changes in the regulation of ciliary beating and proinflammatory cytokine release are enhanced under conditions of alcohol and cigarette smoke coexposure as compared with individual exposures (Elliott et al., 2007). In addition, we have found that only alcohol and cigarette smoke coexposure results in AA and MDA concentrations sufficient for and leading to MAA adduct formation in lung (McCaskill et al., 2011). Furthermore, we identified surfactant protein D (SPD) to be one such adducted protein formed in the lung in response to dual smoke and alcohol exposure (McCaskill et al., 2011). Direct exposure of purified MAA-adducted protein to bronchial epithelial cells in vitro results in a protein kinase C epsilon (PKCɛ)-dependent release of proinflammatory cytokines (Wyatt et al., 2001). We therefore hypothesized that purified MAA-adducted protein would produce inflammatory injury in and of itself when directly instilled into the lungs of mice.

Section snippets

Preparation of MAA-adducted protein

Human lung surfactant proteins were purified from human pulmonary alveolar proteinosis fluid as previously reported (Strong et al., 1998). The MAA-adducted proteins were generated by incubating 1 mg each of bovine serum albumin (BSA) (Fraction V, fatty acid free, low endotoxin; Sigma, St. Louis, MO) and SPD or SPA with an equimolar (1 mmol/L) solution of MDA and AA (Aldrich Chemical Co, Milwaukee, WI) for 3 days at 37°C in a sealed polypropylene vessel in a nonoxidizing atmosphere as detailed in

Surfactant proteins can be MAA adducted

To determine if the biologically relevant lung proteins could form protein adducts with MDA and AA, purified SPA and SPD were reacted in vitro with 2 moles of MDA and 1 mole of AA (Fig. 1). The resulting concentration of MAA-adducted proteins formed was then compared with the amount of MAA that would adduct to BSA. Both BSA and SPD formed MAA adducts (40–50 nmol/mg protein) in equivalent concentrations with no significant differences to each other (Fig. 2). Although the MAA adduction of SPA was

Discussion

In this study, we demonstrate that exposure to MAA-adducted proteins increases lung inflammation in an in vivo mouse exposure model using direct nasal instillation. As evidenced by histology and BAL cell differentiation, this elevated inflammation primarily consisted of a peribronchiolar infiltration of neutrophils in response to nasal instillation with MAA-adducted protein. Mechanistically, this neutrophil recruitment was preceded by MAA-adducted protein stimulation of both epithelial cell

Acknowledgments

This material is the result of work supported with resources and the use of facilities at the Omaha VA Medical Center, Omaha, NE (Department of Veterans Affairs [VA Merit Review] to TAW.) This work was supported by National Institutes of Health–National Institute on Alcohol Abuse and Alcoholism (NIH-NIAAA) (R37AA008769) to JHS, NIH-NIAAA (R01AA017993-S1) to TAW, and NIH-NIAAA (R01AA017993) to TAW.

References (29)

  • D.M. Bowdish et al.

    Conserved domains of the class A scavenger receptors: evolution and function

    Immunol. Rev.

    (2009)
  • Vital signs: current cigarette smoking among adults aged >or=18 years—United States, 2009

    Morb. Mortal. Wkly. Rep.

    (2010)
  • M.J. Duryee et al.

    Scavenger receptors on sinusoidal liver endothelial cells are involved in the uptake of aldehyde-modified proteins

    Mol. Pharmacol.

    (2005)
  • M.K. Elliott et al.

    Effects of cigarette smoke and alcohol on ciliated tracheal epithelium and inflammatory cell recruitment

    Am. J. Respir. Cell Mol. Biol.

    (2007)
  • Cited by (0)

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