br Conclusion br Conflict of interest

Conclusion
Conflict of interest
Acknowledgements
Introduction Human salivary aldehyde dehydrogenase (hsALDH) (E.C. 1.2.1.5) is the first line of defence against toxic aldehydes in the oral cavity. HsALDH is primarily a dimeric, class 3 ALDH (ALDH3A1) specific for aromatic and long/medium chain aliphatic aldehydes as the substrates [[1], [2], [3], [4]]. It is a phase II drug-metabolizing enzyme involved in the detoxification process [5]. It detoxifies the aldehydes in the oral cavity that are either generated endogenously through metabolism or from the exogenous sources such as foods, pollutants, drugs, etc. [6]. These aldehydes are toxic and may cause cancer in the oral cavity if not detoxified [[7], [8], [9]]. Thus, activity of hsALDH in the oral cavity is important to maintain the oral health. It has been reported that the lower activity of this enzyme in the oral cavity is a risk factor for oral cancer development [6,10]. The activity of hsALDH increases during the initial stage of cancer in response to the cancer development and decreases after the surgical removal of tumor, indicating that cancinogenesis induces the ALDH enzymes [10,11]. The over expression of ALDHs in cancerous tissues results in increased resistance to oxazaphosphorine based chemotherapy [12]. HsALDH activity is affected by ethanol, hydrogen peroxide and sodium dodecyl sulfate under in vitro conditions [3]. ALDHs have been found to be induced by various aryl and aromatic hydrocarbons [13]. ALDH activity and expression was reported to be induced in the oral cavity upon exposure to cigarette smoke and tobacco products [4,14,15]. The enzyme protects the oral epithelial 2881 from the cigarette smoke induced cytotoxicity and DNA damage. HsALDH activity was also observed to be altered by different dietary components, such as vegetables and fruits. Intake of large quantities of coffee and vegetables like broccoli were found to activate and induce the enzyme in the saliva of human subjects [16]. It has been recently reported that the active component of broccoli i.e., sulforaphane, and of black cumin seeds i.e. thymoquinone directly activate the enzyme [17,18]. Moderate consumption of coffee was found to decrease the hsALDH activity, whereas consumption of large quantities of coffee increases its activity in the saliva. It has been speculated that the former is due to the inhibition of hsALDH by some coffee component, and the latter is due to induced expression of the enzyme, which is probably a defence mechanism in response to the lowered activity of the enzyme [14,16]. Caffeine, theophylline and related compounds have been reported to inhibit the activity of salivary ALDH [19]. The activity of hsALDH in saliva is crucial for maintaining the oral health and prevention of aldehyde mediated toxicity [10,20]. Therefore, the effect of dietary components frequently encountered by the enzyme, on its activity is important to study for nutrition safety and health [10,14]. Caffeine is an alkaloid present in food items, beverages, coffee, tea, etc. Chemically, it is 1,3,7-trimethylpurine-2,6-dione (Fig. 1). It is frequently taken in large quantities on a regular basis around the globe [21,22]. The caffeine content in various food items ranges from 2 to 200 mg/150 ml [21]. A Canadian survey has shown that caffeine content in coffee/tea varies from 30 to 150 mg/cup [21]. The quantity of caffeine consumed through beverages among the U.S. population varies from 225 to 400 mg/day [23]. Small quantity of caffeine intake has been found to be safe and beneficial for health, whereas consumption of large quantity of it causes several adverse health effects [22]. Since caffeine affects the activity of ALDH in the saliva, therefore, a detailed account of its effect on the enzyme kinetics, mechanism of inhibition and interaction with the enzyme is important to examine. In the present study, we have reported the effect of caffeine on the activity (both dehydrogenase and esterase activity) and kinetics of hsALDH. Further, the mechanism of inhibition of caffeine and its binding with hsALDH was also been studied using different biophysical techniques and molecular docking analysis.