Project Summary/Abstract. Electronic cigarettes are gaining popularity as alternative to traditional cigarettes, with sales increasing from $283 million in 2012 to $2.5 billion in 2018 in the US. The global e-cigarette market is estimated to reach $24.2 billion by 2024. E-cigarette solutions, also known as e-liquids, are highly variable with enticing flavors, such as tobacco, menthol, fruit, candy, and dessert. Liquid nicotine concentrations vary in these products from 0 to 100 mg/ml according to an FDA study. Although nicotine toxicity in adults is rare with an estimated lethal dose between 60 and 500 mg (0.8-6.7 mg/kg), flavored e-liquids are increasingly being ingested orally, putting children at risk for exposure to high concentrations of nicotine. Indeed, nicotine toxicity in children under 5 years of age can occur with consumption of as little as a teaspoon of liquid nicotine. The unforeseen consequences of e-cigarettes, with respect to nicotine poisoning in children, presents an unmet need to counteract the harmful and potentially fatal outcomes that may occur among this vulnerable population. It is the pediatric population we plan to address in this proposal, as a high potential of accidental ingestion of liquid nicotine from e-cigarettes exists. Despite the increase in nicotine-related poisonings reported, there is no treatment for acute nicotine toxicity. Current treatment regimens for nicotine poisoning range from supportive care, to activated charcoal, to respiratory support with mechanical ventilation. An alternative means of altering the toxicity of nicotine poisoning could come via simple sequestering of the drug. Antibodies to nicotine have been prepared as a means to block the pharmacological effects of this drug. To date, vaccines for smoking cessation have shown promise in preclinical animal models; however, in clinical studies, these vaccines failed to measure significant differences in smoking abstinence between the intervention and placebo groups. Thus, the likelihood of an antibody attenuating an acute dose of nicotine is doubtful. What is needed is a sufficient pharmacokinetic (PK) biologic with the capacity to not just sequester nicotine but also increase its metabolism. We envision a biologic able to catabolize nicotine rather than simply sequestering the drug would have the potential to treat acute nicotine poisoning. The proposal at hand details a bacterial strain, Pseudomonas putida, which has evolved to use nicotine as its sole source of carbon and nitrogen. From this bacterial strain, we will examine a first-in-class enzyme, a nicotine oxidoreductase termed NicA2, as a means to treat nicotine poisoning. Our initial characterization of the enzyme indicates that it could be an excellent candidate for altering nicotine poisoning. However, the successful demonstration of this enzyme reversing nicotine poisoning will require several experimental undertakings including: (1) Evaluating the efficacy of NicA2 to attenuate ac...