Acute Acrolein Exposure Induces Impairment of Vocal Fold Epithelial Barrier Function

Abstract

Acrolein is a ubiquitous pollutant abundant in cigarette smoke, mobile exhaust, and industrial waste. There is limited literature on the effects of acrolein on vocal fold tissue, although there are clinical reports of voice changes after pollutant exposures. Vocal folds are responsible for voice production. The overall objective of this study was to investigate the effects of acrolein exposure on viable, excised vocal fold epithelial tissue and to characterize the mechanism underlying acrolein toxicity. Vocal fold epithelia were studied because they form the outermost layer of the vocal folds and are a primary recipient of inhaled pollutants. Porcine vocal fold epithelia were exposed to 0, 50, 100, 500, 900 or 1300 μM of acrolein for 3 hours; the metabolic activity, epithelial resistance, epithelial permeability, tight junction protein (occludin and claudin 3) expression, cell membrane integrity and lipid peroxidation were investigated. The data demonstrated that acrolein exposure at 500 μM significantly reduced vocal fold epithelial metabolic activity by 27.2% (p≤0.001). Incubation with 100 μM acrolein caused a marked increase in epithelial permeability by 130.5% (p<0.05) and a reduction in transepithelial electrical resistance (TEER) by 180.0% (p<0.001). While the expression of tight junctional protein did not change in acrolein-treated samples, the cell membrane integrity was significantly damaged with a 45.6% increase of lipid peroxidation as compared to controls (p<0.05). Taken together, these data provide evidence that acute acrolein exposure impairs vocal fold epithelial barrier integrity. Lipid peroxidation-induced cell membrane damage may play an important role in reducing the barrier function of the epithelium.

Fig 1. The metabolic activities of vocal fold tissue following acrolein exposure ex vivo.

Epithelial metabolic activity was determined by the MTT assay. Tissues were incubated with acrolein as the concentration indicated for 3 hours. Data represent Mean ± SE, n = 7, *: p < 0.05, §: p < 0.001 as compared to controls.

Fig 2. Barrier permeability assay with or without acrolein exposure.

Tissues were incubated with acrolein as the concentration indicated for 3 hours. (A). The TEER values were assessed by Ussing chamber system and associated voltage clamp. (B). The epithelial permeability was measured by fluorescent marker NaFI. Index of permeability represents the percentage of fluorescent marker passing though epithelium. Data represent Mean ±SE, n = 7, *: p < 0.05 as compared to controls.

Fig 3. Expression levels of mRNAs encoding typical tight junctional proteins.

Tissues were incubated with acrolein as the concentration indicated for 3 hours. The mRNA levels of occludin (A) and claudin3 (B) were determined by qPCR. Data represent Mean ± SE, n = 6 (p = 0.259 for occludin and p = 0.556 for claudin3) as compared to controls.

Fig 4. Western blot analysis of occludin protein with or without acrolein exposure.

Data represent Mean ± SE, n = 6, p = 0.337 as compared to controls. “C” represents control group and “A” represents acrolein treated group.

Fig 5. Cell membrane integrity assessed by LDH leakage.

Data represents LDH release in extracellular medium normalized by the average in the control group, #: p<0.01 as compared to controls by paired t-test.

Fig 6. Immunohistochemical study of lipid peroxidation marker with or without acrolein exposure.

(A). A typical confocal image of vocal fold epithelia. 4-HNE was stained in red on the left panel; occludin was stained in green on the middle panel; and the merged signals in yellow was present on the right panel. (B). Quantification of signal intensities of 4-HNE and occludin. Data represent Mean ± SE, n = 4, *: p < 0.05 as compared to controls.