Thymoquinone binds and activates human salivary aldehyde dehydrogenase: Potential therapy for the mitigation of aldehyde toxicity and maintenance of oral health
Introduction
Human saliva contains many detoxifying and antioxidant enzymes like glutathione S-transferase, catalase, peroxidase, aldehyde dehydrogenase (ALDH), etc. [1]. Human salivary ALDH (hsALDH) protects individuals from toxic aldehydes contained in food as natural ingredients, preservatives or contaminants, or even those produced during lipid oxidation, as well as those generated by inhalation of cigarette smoke, pollutants and drugs [2]. HsALDH is mainly a single, dimeric isoenzyme, belonging to class 3 ALDH and classified as ALDH3A1 (EC 1.2.1.5) [3]. It is an enzyme of broad substrate specificity preferential for long/medium chain aliphatic aldehydes and aromatic aldehydes including toxic 4-hydroxy-2-nonenal, but is inactive towards acetaldehyde [4]. The best substrates of hsALDH belong to the aromatic group which are cinnamic aldehyde, benzaldehyde and anisaldehyde, both in terms of Km and Vmax/Km [4]. It undergoes reversible oxidation in thiol free medium. Dithiothretiol (DTT) or dithioerythritol are used to regenerate the oxidized enzyme in vitro [5].
The activity of hsALDH is highly variable in the healthy population, and depends on many factors such as age, cigarette smoking, alcohol consumption, pollution, diet, drug consumption, etc. [6], [7]. Lower activity of hsALDH results in reduced protection of the oral cavity against oxidative stress, which may make the individual susceptible to carcinogenesis [8]. Inducers of this enzyme has shown to prevent experimental carcinogenesis [2]. Individuals who ingests large amounts of coffee and broccoli were found to have elevated level of this enzyme in their saliva [7]. Many strategies have been employed to restore the activity of mutated ALDH, enhance the activity in vitro or to induce the enzyme level in vivo [9], [10], [11], [12], [13]. Specific activators of ALDH such as Alda-89, Alda-1, tamoxifen, etc., have been studied which have shown impressive results in mitigating ALDH related pathogenesis in cell lines and in animal models [9], [14], [15], [16]. Therefore, the knowledge about factors influencing the hsALDH activity seems to be important for food and drug safety as well as for nutritional research [7], [17].
The Nigella sativa (black cumin) is an annual flowering plant which has been frequently used in folk medicine for the treatment of various diseases for centuries, both as an herb as well as pressed into oil [18], [19]. It is used to treat ailments such as asthma, bronchitis, rheumatism and to fight parasitic infections and many other diseases [20]. Thymoquinone (TQ), 2-isopropyl-5-methylbenzo-1,4-quinone is the main active and the most abundant constituent of the volatile oil of the cumin seeds [21]. It has been shown to have many beneficial pharmacological effects such as anti-oxidant, anti-inflammatory, immunomodulatory, anti-microbial, anti-diabetic, anti-tumor, hepatoprotective, neuroprotective and gastroprotective effects, etc. [22], [23], [24], [25], [26]. TQ has been found to exhibit promising anti-carcinogenic, anti-neoplastic, anti-proliferative, anti-mutagenic and apoptosis inducing activities against various tumor cells [27], [28].
Many chronic diseases are caused by an alleviated level of oxidative stress in the body. To combat this, wide arrays of cytoprotective factors are activated. TQ has been reported to induce activation of these cytoprotective proteins, and hence it is involved in the cellular anti-oxidant defence or inactivation of electrophilic carcinogens, thus preventing oxidative stress [29]. Its anti-oxidative potential is related to the redox properties of the quinone molecule, unrestricted mobility to cross physiological barriers and easy approach to subcellular compartments [30]. The cumin seeds and TQ have been found to maintain the oral health [31]. They mitigate the toxicity induced by anticancer drug cyclophosphamide [32]. TQ has been shown to have a positive effect on the lipid peroxidation level, activity of antioxidant enzymes and also ameliorates the oxidative stress [33], [34]. Therefore, physiological function of hsALDH and the pharmacological activities of TQ in vivo are similar. If TQ is found to have a direct effect on the activity of hsALDH in vitro, its pharmacological effects and mechanism of action can be established.
HsALDH enzyme has been purified for the first time in our laboratory from human saliva and has been kinetically characterized using different aromatic substrates [35]. Also, the effect of some common substances frequently encountered by the enzyme in the oral cavity (such as ethanol, hydrogen peroxide and sodium dodecyl sulfate), on its activity has been investigated [35]. More recently, the activity and stability of the purified enzyme was revealed under different conditions such as temperature, in presence of denaturants and salt [36]. Further, the storage stability of the enzyme was determined under in vitro conditions and in presence of stabilizing agents [36]. In one of our previous study, a small molecule chemical activator, Alda-1 was designed for ALDH2 which activated the enzyme and restored the activity of its inactive mutated form i.e., ALDH2*2 [11]. Recently, we have also reported sulforaphane, a natural molecule found in cruciferous vegetables as an activator of hsALDH, which increased the dehydrogenase activity of the enzyme by almost two fold [37]. The present study aimed at searching for a better natural molecule activator of hsALDH from various sources and found TQ as a suitable candidate. Therefore, we have studied the effect of TQ on the activity (dehydrogenase and esterase activity) and kinetics of both crude and purified hsALDH. The binding of TQ with the pure hsALDH was studied using different biophysical techniques such as UV–vis, fluorescence, CD spectroscopy and FRET analysis. Molecular docking was performed to determine the binding site, amino acid residues involved and the type of interactions between TQ and hsALDH. The interaction of TQ with hsALDH and its effect on the enzyme activity is expected to have a great importance from the point of view of aldehyde related pathogenesis, carcinogenesis and nutritional benefits on health.
Section snippets
Materials
TQ, 6-methoxy-2-naphthaldehyde, 6-methoxy-2-naphthoic acid, NAD+, bicinchoninic acid (BCA) and bovine serum albumin (BSA) were purchased from Sigma Chemicals Co., USA. Diethylaminoethyl-cellulose (DEAE-cellulose), dimethyl sulphoxide (DMSO), dithiothreitol (DTT), p-nitrophenyl acetate (pNPA), 2-morpholinoethane sulphonic acid (MES) and acetonitrile were from Sisco Research Lab., India. Di-sodium hydrogen phosphate, sodium phosphate monobasic anhydrous, EDTA, NaCl, acetic acid and Tris were
Effect of TQ on the dehydrogenase activity of hsALDH
The effect of TQ on the activity of crude and purified hsALDH was studied. It was found that the activity increased with the increase in TQ concentration for both crude (Fig. 1A) and purified enzyme (Fig. 1B) upto 250 nM, beyond which the activity became almost constant. At 250 nM TQ, about 3.2 and 2.9 fold increase in the initial velocity was observed for the crude and the pure enzyme, respectively. The concentration of TQ at half maximum response (EC50 value) was determined to be 103.6 ± 2.5 nM
Discussion
In one of our previous studies, we have reported a small molecule activator (Alda-1) of the wild type ALDH2 which also restored the activity of the inactive, mutated ALDH2*2 [11]. More recently, we have found sulforaphane from cruciferous vegetables as an activator of hsALDH, having significance in the metabolism of acetaldehyde [37]. It activated the enzyme by almost two fold. In the present study, we report the natural compound, TQ as a better activator of hsALDH, which increased the
Conclusion
TQ activated hsALDH by enhancing its substrate binding affinity and catalytic efficiency. Biophysical investigations revealed that TQ forms a complex with hsALDH in its active site and this interaction is responsible for the activation of the enzyme. The activation of hsALDH activity is of relevance from the point of view of oral health and protection from oral cancer. TQ is therefore expected to give better protection from the toxic aldehydes in the UADT including oral cavity and the risk of
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
Facilities provided by the Aligarh Muslim University are highly acknowledged. AAL is thankful to the Department of Biotechnology, Govt. of India for providing fellowship in the form of SRF.
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