Tobacco smoke is the primary cause and very often exacerbates many respiratory diseases.Cigarette smoke results in chronic obstructive pulmonary diseases,asthma,decline in lung function,pulmonary irritation,allergy,infections,and impaired lung development in children.Of 7,357 tobacco constituents,at least 250 are harmful,and more than 69 are carcinogenic.However,the threshold values of the respiratory toxic constituents are unknown.The airways and alveolar sacs are lined with an epithelial cell layer,the first defense against the inhaled smoke toxicants.The epithelial sodium channels(ENaC)located in the cilia control airway fluid homeostasis.Tobacco smoke and extracts altered ENaC,we aimed to examine whether ENaC could be a functional biomarker for screening smoke constituents irritant to the respiratory system in a real time and paired manner.We evaluated 20 constituents recommended by the FDA in an expression system,human airway epithelial cells,and in mice.Our results show that ENaC activity was acutely reduced by up to 80%by three constituents(crontonaldehyde,formaldehyde,and acrolein)in minutes.By contrast,carbon monoxide and nicotine transiently stimulated ENaC(P<0.05).Eleven of 20 constituents increased the ion influx across the plasma membrane,indicating severe injury in the integrity of cell walls.Acrolein(80 Bg)reduced ENaC activity in primary epithelial cells and in vivo significantly following a 30-minute exposure.The safety level for acrolein was approximate 0.001 Bg per cigarette.These data demonstrate that ENaC function and ion permeability are novel biomarkers for effectively evaluating tobacco toxicity.Tobacco aldehydes are potent toxicants to ENaC function.The high throughput oocyte model will facilitate the screening of potentially harmful 7357 constituents.The thresholds of each harmful constituent and their mixtures will provide direct evidence for preparing the guidelines regarding the safety level in tobacco products and for public exposure.