PROJECT SUMMARY/ABSTRACT Psychoactive substances provide temporary feelings of euphoria, but also elicit multifold harm to the human body. Areca (betel) nut is the 4th most common abused substance worldwide, only behind alcohol, caffeine, and nicotine. Though AN chewing is traditionally recognized abroad, emerging evidence demonstrates use within migrant and refugee communities in the US. Despite global popularity, there is a dearth of knowledge about how the predominant psychoactive and toxic substance in the nut, arecoline, is metabolized and the biological consequences if its metabolism is perturbed by other substances. Ethanol is commonly co-abused with AN, and significantly their combined ingestion is associated with greater patient harm for reasons yet to be determined. Our supporting data demonstrate that ethanol inhibits arecoline metabolism, suggesting arecoline accumulation will occur in the blood and brain tissue – a highly significant toxicokinetic interaction. We have also identified that L-menthol is present in commercialized (branded) AN products sold in the US, and our preliminary findings show menthol deters arecoline hydrolysis. Taken together, the long-term goal of this project is to improve the health of AN substance abusers. The overall objectives in this application are to (i) confirm human hydrolase(s) and tissue site(s) catalyzing hydrolysis of arecoline; (ii) characterize a newfound ethanol drug interaction with arecoline; and (iii) distinguish changes in arecoline's pharmacokinetic profile and neurobiological activity following co-ingestion with L-menthol, a substance of emerging concern in branded AN products. The central hypothesis is that arecoline is subject to metabolic inhibition when combined with ethanol or L-menthol leading to increased toxicity and greater CNS effects. The central hypothesis will be tested by 3 specific aims: 1) Establish the predominate contribution of human esterases and tissue sites that hydrolyze arecoline; 2) Dissect the influence of ethanol on arecoline disposition; and 3) Elucidate the pharmaco- neurological significance of menthol in commercialized AN products. The aims will be accomplished by an interdisciplinary team with varied expertise in trace drug analysis, carboxylesterase drug metabolism, biomimetic 3D organoid systems, special knock-out mouse models, and drug addiction. Male and female mice will be used throughout the aims to assess sex as a biological variable. Ultimately after 5-years, we expect to be the first to demonstrate arecoline is predominantly metabolized in the liver by carboxylesterase-1 (CES1), an enzyme we anticipate catalyzes transesterification of arecoline in the presence of ethanol, forming a unique metabolite with toxicological properties. Further, we anticipate L-menthol combined with arecoline will provoke arecoline buildup in brain tissue and modify the neurobiological profile and psychoactivity in mice, a brand new pharmaco-neurological interaction. Fin...