A Review on Role of Arecoline and Its Metabolites in the Molecular Pathogenesis of Oral Lesions with an Insight into Current Status of Its Metabolomics

Abstract: Areca nut consumption is a popular habit in Southeast Asian countries. One of the important biologically active alkaloids of areca nut is arecoline, which plays a role in mediating the development of several pathologies of the primary exposure site, the oral cavity. Studies on the metabolism of arecoline revealed the formation of several metabolites which themselves might be toxic. Moreover, polymorphisms in genes encoding enzymes involved in the metabolism of arecoline might predispose an organism towards the development of oral cancer. The present review tries to accumulate all the relevant existing literature and then elucidate the molecular mechanism by which arecoline plays a role in the development of oral submucous fibrosis and oral cancer. Existing information regarding arecoline metabolism, enzymes involved in the metabolic process and biological effects of the metabolites of arecoline have also been compiled and compared to study the toxicity of metabolites with its parent compound arecoline and whether they play any role in the pathogenesis of oral cancer mediated by areca nut consumption. A repertoire of molecular targets has come up in the discussion whose expression profile is perturbed by arecoline. Construction of induction cascade from existing literature has given an idea about the process of molecular pathogenesis. The summarized and analysed data can help to determine the molecular mechanism and drug targets,


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(4) understand the mechanism of metabolism of arecoline and arecoline N-oxide by gathering knowledge about enzymes involved, which in turn may enable to estimate predisposition of an individual towards the development of oral cancer in areca nut consumers.

Metabolism of arecoline
The metabolic profile of arecoline might play a role in areca nut mediated pathogenesis of OSF and OSCC. This brings out the need to delve into the depths  N-nitrosonipecotic acid. (b, c, d, e, h, i, j, k, l, m, p, q, r -metabolites of arecoline; d, e, h, i, j, l -metabolites of arecaidine; a, b, f, g, k, m, n, o -metabolites Report / Vol. 121 (2020) No. 4, p. 209-235 Figure 2 -Metabolic map of arecoline and its metabolites. Pathway adapted from Wenke and Hoffman (1983), Ohshima et al. (1989), Miyazaki et al. (2005), Giri et al. (2006Giri et al. ( , 2007. The symbol "?" represents the unavailability of knowledge about the enzyme involved. Enzyme involved in each step has been mentioned in italics.

Arecoline and Metabolites: Role in Oral Pathologies
Prague Medical Report / Vol. 121 (2020) No. 4, p. 209-235 213) of the metabolomics of arecoline and explore its potential towards the development of oral pathology. Metabolism of arecoline starts in the oral cavity itself. Both nitrite and thiocyanate (catalyst for nitrosation reaction) are present in human saliva (Boyland et al., 1971;Shivapurkar et al., 1980;Wenke et al., 1984a). When arecoline was incubated with sodium nitrite with or without sodium thiocyanate, the formation of three compounds was observed: 3-(methylnitrosamino)propionaldehyde (MNPA), 3-(methylnitrosamino)propionitrile (MNPN) and N-nitrosoguvacoline (NGL) (Wenke and Hoffmann, 1983). Of these, NGL and MNPN were detected in the saliva of betel quid chewers (without tobacco) in the range of 2.2-9.5 µg/l and 0.5-11.4 µg/l, respectively (Wenke et al., 1984a;Prokopczyk et al., 1987). These findings indicate that nitrosation of arecoline does take place in the oral cavity of areca nut consumers, and thereby, buccal cells do get exposed to these nitrosated metabolites on chewing areca nut. In a mammalian test system, N-nitrosonipecotic acid (NNIP) was detected as a product of metabolism of NGL (Ohshima et al., 1989). MNPA has not been detected in saliva samples of areca nut consumers. However, as the formation of MNPA from arecoline takes place under in vitro conditions, fast metabolism of MNPA to its metabolites in vivo might explain this finding. The metabolome of arecoline, arecaidine and arecoline N-oxide in vivo was investigated by Nery (1971) and Giri et al. (2006Giri et al. ( , 2007. This research revealed several unknown and novel metabolites ( Figure 1). Of these, arecaidine and N-methyl nipecotic acid were detected in the urine of areca nut consumers (Hu et al., 2010). Figure 2 depicts a metabolic map of arecoline.

Role of arecoline in induction of oral pathologies
Genotoxic effects of arecoline were proved in mice using tests evaluating the formation of chromosomal aberrations and micronucleus (Shirname et al., 1984;Deb and Chatterjee, 1998). At the molecular level, arecoline induces a DNA damage response cascade involving phosphorylation of ataxia-telangiectasia (ATM) kinase and its downstream targets checkpoint kinase 1/2 (Chk1/2), p53 and Nbs1, leading to a G2/M cell cycle arrest. However, the overall expression of p53 is downregulated by arecoline, which is followed by suppression of p53 mediated DNA repair activities and expression of its downstream target p21 WAF1 . Arecoline has also been found to induce decreased expression of p21 and p27 via a p53 independent process that includes reactive oxygen species (ROS) and activation of mammalian target of rapamycin complex-1 (mTORC1) pathway ( Ji et al., 2012). According to Ji et al. (2012), down-regulation of these inhibitors of cell cycle might lead to erroneous DNA replication as the cells escape the G1/S checkpoint. Arecoline also disturbs the fluidity of polymerisation-depolymerisation kinetics of α-tubulin by favouring their polymerisation. It leads to a disfigurement of the mitotic spindle and an erroneous arrangement of chromosomes, thereby inducing the pro-metaphase cell cycle arrest . Apart from these alterations,

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protein expression of several other cell cycle regulatory molecules like cdc25c in basal carcinoma cells , cyclin B1 and Wee-1 in KB epithelial cells (Lee et al., 2006) and cyclin D1, cyclin A, cyclin E, CDK4, and CDK2 in HaCaT keratinocytes (Zhou et al., 2013) have been found to be modulated by arecoline. Arecoline treatment leads to the down-regulation of the immune system in mice (Dasgupta et al., 2006;Wen et al., 2006). By contrast, Hung et al. (2011) reported the production of ROS in endothelial cells on exposure to arecoline, which resulted in an up-regulated expression of adhesion molecules (intercellular adhesion molecule -ICAM, and vascular cell adhesion molecule -VCAM). This effect increased adhesion between mononuclear cells and endothelial cells, which might play a role in augmenting the inflammation. Additionally, several inflammatory cytokines have also been found to be induced by arecoline, such as interleukin-1α, prostaglandin E-2, and cycloxygenase-2 in fibroblasts ( Jeng et al., 2003;Tsai et al., 2003;. DNA damage and impaired DNA repair, along with chronic inflammation, can be emphasized as the main causes of arecoline induced oral pathologies. Moreover, expression profiles of several extracellular matrix (ECM) proteins, enzymes, growth factors, and transcription factors are altered under the effect of arecoline ( Figure 3). All these factors work in an association as indicated by several studies involving fibroblasts, epithelial cells and cancer cell lines (Tables 1 and 2).
Oral submucous fibrosis (OSF) is a pre-cancerous condition that develops from an abnormal wound healing process under continuous exposure to the components of areca nut (Angadi et al., 2011). Inhibition of elements involved in the degradation of extracellular matrix (ECM) or enhanced stability and synthesis of matrix components disturbs the homeostasis of ECM, which can give rise to disease conditions like fibrosis. Arecoline works positively in both these aspects. It enhances the expression of several inhibitors of proteinases, including tissue inhibitors of metalloproteinases (TIMPs), plasminogen activator inhibitors (PAI-1) and cysteine proteinase inhibitor cystatin C in fibroblasts (Chang et al., 2002a;Yang et al., 2003;Tsai et al., 2007) along with induction of factors that enhance the stability of ECM, such as heat shock protein-47 (Hsp-47) and transglutaminase-2 (TGM-2) Lee et al., 2015). Moreover, arecoline has been found to decrease the phagocytosis of collagen by fibroblasts (Shieh et al., 2004).
An analysis of the repertoire of moieties affected by arecoline (data compiled in Tables 1 and 2) has bought into light several mediators of the effects induced by arecoline of which three, ROS, transforming growth factor-β1 (TGF-β1) and hypoxia-inducible factor-1α (HIF-1α), might play key roles in the pathogenesis of OSF and OSCC.
Reactive oxygen species involved in induction. Moderate/advanced OSF specimen showed higher expression of transglutaminase-2 in fibroblasts.
Another important mediator of arecoline induced effects is HIF-1α. Apart from being induced by arecoline itself in fibroblasts and epithelial cells Tsai et al., 2015), it plays a part in the induction of several downstream factors that overlaps the repertoire induced by TGF-β1 ( Figure 3) (Higgins et al., 2004;Tsai and Wu, 2012). Hypoxia prevails in fibrotic as well as tumour conditions, which prevents degradation of HIF-1α. Under the hypoxic condition, stabilized HIF-1α dimerizes with HIF-1β and, in association with co-activators, it participates in the transcription of genes with hypoxia-responsive element (HRE) (Tsai and Wu, 2012;Eckert et al., 2016;Rankin and Giaccia, 2016). As reviewed by Tsai and Wu (2012) and Rankin and Giaccia (2016), several genes involved in metastasiring are regulated by HIF-1α, including transcription factors involved in EMT (Twist, Snail, Aeb1/2, etc.), enzymes like matrix metalloproteinases (MMP 1/3), matricellular proteins (cysteine rich protein 61 [Cyr61]), and angiogenic factors (vascular endothelial growth factor).
Several of the pro-fibrotic or carcinogenic factors induced by arecoline are coupled with a decrease in intracellular thiol content and show reversible expression after treatment with antioxidants (Tables 1 and 2). This emphasizes ROS generation by arecoline to be a "cause" of the various arecoline driven "effects" that trigger OSF or OSCC. Interplay can be observed between ROS and TGF-β1 as well as HIF-1α. For instance, ROS plays a part in the activation of latent TGF-β1 complex tethered to the ECM ( Jobling et al., 2006). In turn, TGF-β1 acts to increase ROS production via the activity of its downstream target NADPH oxidase-4 (NOX-4) along with suppression of the antioxidant defence system of exposed cells (Richter and Kietzmann, 2016). Apart from sharing a common array of downstream targets, TGF-β1 and HIF-1α augment each other's expression too. HIF-1α supports transcription of TGF-β1 under hypoxic conditions, whereas, under normoxic conditions, TGF-β1 enhances the stability of HIF-1α by decreasing expression of HIF-1α inhibitor prolyl hydroxylase 2 (PHD2) (McMohan et al., 2006;Hung et al., 2013). ROS induces HIF-1α stability and thereby its transcriptional activity via an adenosine monophosphate-activated protein kinase (AMPK) pathway ( Jung et al., 2008). AP-1, ERK, PKC pathway. Decreased intracellular thiol content involved in induction. Higher expression was seen in OSCC cases of poor differentiation. In between OSCC specimen, association found between low grade expression and lymph node metastasis. Lee et al. (2008a) 3.
Decreased intracellular thiol content involved in induction. Higher expression was seen in OSCC cases.
ERK, Cox-2, PI3K, tyrosine kinase pathways. Decreased intracellular thiol content involved in induction. Higher expression was seen in OSCC cases. In between OSCC specimen, association found between low grade expression and lymph node metastasis. Lee et al. (2011) 6.
Cox-2, ERK, p38 pathways. Decreased intracellular thiol content involved in induction. Higher expression was seen in OSCC cases. In between OSCC specimen, association found between low grade expression and lymph node metastasis.

Higher expression is seen in cases of OSCC samples.
Association found with lymph node metastasis and poor differentiation of OSCC samples. -Higher expression of ZEB1 mRNA was seen in recurrent cases of OSCC than in initial stages.
-Higher expression of Lin28B mRNA is seen in cases of OSCC samples. Association found with lymph node metastasis.
Lin et al.
PI3K, JNK pathway. Hypoxia inducible factor 1α involved. Higher expression seen in cases of OSCC samples, especially those that were moderately or poorly differentiated. Association found with lymph node metastasis.

Hu et al. (2015)
LT -long-term; ROS -reactive oxygen species; PI3K -phosphatidylinositol 3-kinase; Cox-2 -cycloxygenase-2; JNK -c-Jun NH ROS involvement in cancer includes induction of pathways like mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3K)/Akt and nuclear factor kappa-light chain enhancer of activated B-cells (NF-κB) (Liou and Storz, 2010). Analysis of the data compiled in Tables 1 and 2 also indicates these pathways to be involved in the up-regulation of pro-fibrotic or carcinogenic factors in cell systems affected by arecoline. Targeting these pathways might be a promising tool in the therapy of ROS induced OSF and OSCC. Figure 4 depicts hypothetical mechanistic pathways where ROS induced DNA damage along with perturbation of expression profile of growth factors, transcription factors and ECM proteins drive the development of areca nut mediated oral pathologies.
As mentioned earlier, OSF can develop into a malignant phenotype. Therefore, it is important to understand how arecoline triggers the expression of effectors that mediate this transition. Under continuous exposure to hypoxic conditions and HIF-1α activity, the transformation from fibrotic to cancer condition might take place, thereby highlighting arecoline's role as a tumour promoter. Arecoline induced stabilisation of HIF-1α and activation of TGF-β1 play roles in the regulation of

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several transcription factors, such as ZEB-1, Snail, Twist, that induce both type 1 and type 2 EMT leading to fibrosis and cancer, respectively Chang et al., 2014;Ho et al., 2015;Lee et al., 2016). Another interesting aspect of arecoline, which was discovered during the study, is its differential activity to induce ECM modulators in fibroblasts and cancer cells. Exposed fibroblasts up-regulate TIMP-1, whereas MMP-2 is inhibited (Chang et al., 2002a). In cancer cells, MMP-9 is induced, but TIMP-1 is inhibited (Chang et al., 2013a). TGF-β1 has been found to regulate the expression of MMP-9 via Snail transcription factor in cancer cells (Sun et al., 2008). After exposure of cells from cancer cell line to arecoline for an extended period of time, increased expression of metalloproteinases MMP-8 and MMP-1 was observed (Liu et al., 2007;Lee et al., 2008). This might indicate that under already initiated and promoted tumour conditions, arecoline might play a role in the progression of areca nut induced carcinogenesis.
Junctional protein disruption is an essential phenomenon by which cells can acquire a metastatic phenotype (Parker et al., 2001). Giri et al. (2010) found arecoline to down-regulate tight junctional protein zona occludens-1 (ZO-1) and claudin-1 along with delocalisation of both ZO-1 and E-cadherin. Down-regulation of E-cadherin during junctional protein disruption leads to the dislocation of β-catenin from the plasma membrane. Under the influence of Wnt signalling in cancer conditions, degradation of cytoplasmic β-catenin is also inhibited. Both junctional and cytoplasmic β-catenin then moves to the nucleus and acquires transcriptional control over genes, leading to abnormal cell proliferative activity (Kam and Quaranta, 2009;Camilli and Weeraratna, 2010;Liu and Millar, 2010). Arecoline exposure induces elevated expression of β-catenin in epithelial cells (Lee et al., 2012b). Signalling cascades have been constructed using the accumulated data in Figure 3.

Role of arecoline metabolites in areca nut induced oral pathologies
Formation of these metabolites in vitro or in in vivo systems indicates the probability of exposure of humans to these metabolites. An assessment of the biological effects of these metabolites is, therefore, necessary to understand the areca nut mediated pathogenesis of OSF or OSCC.
The N-nitrosated metabolites, NMPA, NMPN and NGL, induce DNA single-stranded breaks in epithelial cells where NMPA was the strongest while NMPN and NGL were weak inducers (Sundqvist et al., 1989). Of the in vivo metabolites, genotoxic effects were displayed by arecaidine in mice via induction of sister chromatid exchanges (SCE) in bone marrow cells (Panigrahi and Rao, 1984). Arecoline N-oxide was also found to be genotoxic in both mice model and fibroblasts (Kuo et al., 2015). The genotoxic potential of the other metabolites of arecoline is unknown.
Both MNPA and MNPN were found to be carcinogenic in rats (Wenke et al., 1984b;Nishikawa et al., 1992). NGL was neither found to be a strong mutagen in the bacterial test system nor a strong carcinogen in the murine test system (Rivenson et al., 1988;Miyazaki et al., 2005). Activated N-nitrosamines can cause alkylation of DNA base pairs (Miyazaki et al., 2005). Hence, activated areca nut derived nitrosamines can initiate carcinogenicity via alkylation of DNA, as indicated by the study where MNPN was observed to induce methylation and cyanoethylation of guanine residues in rats, especially in the genetic material obtained from nasal mucosa, esophagus and liver (Prokopczyk et al., 1987. The role of arecaidine as a profibrotic agent remains unclear as indicated by contradictory reports in in vitro models (Harvey et al., 1986;Tsai et al., 1999;Chang et al., 2013b).
The most important metabolite of arecoline might be arecoline N-oxide. It was found to be mutagenic in bacteria without any metabolic activation (Lin et al., 2011). In mice, the compound induced increased collagen deposition in the tongue along with hyperplasia. Several pro-fibrotic genes (TGF-β1, IL-6, S100A4, and fibronectin) were induced by the compound in fibroblasts along with suppression of E-cadherin (Kuo et al., 2015). Therefore, fibrosis induced by areca nut chewing can be mediated partially by arecoline N-oxide. In another study, N-oxide induced sub-lingual hyperplastic lesions in mice along with the up-regulated expression of caspase-8, which, instead of producing a pro-apoptotic effect, enhanced cell survival and proliferation (Ko et al., 2018). 8-hydroxydeoxy guanosine level in fibroblasts cultured in the presence of arecoline N-oxide indicates oxidative stress induced DNA damage (Kuo et al., 2015). Similarly to arecoline, supplementation of thiol-containing agents can reverse the mutagenic property of the compound (Lin et al., 2011). These facts indicate oxidative stress to be an important factor for both arecoline and arecoline N-oxide induced pathologies. Hence, the consumption of antioxidants can be a preventive factor against the development of fibrosis and areca nut driven oral cancer. Table 3 summarizes the various effects of arecoline metabolites. It also provides an assessment of the potential harmfulness of the metabolites based on the possibility of involvement in areca nut induced pathologies. Although arecoline N-oxide has been discovered as a possible candidate mediating areca nut effects, in the in vivo studies mentioned above, arecoline N-oxide was administered via oral brushing. On the other hand, Lin et al. (2011) observed that the compound lost its mutagenicity after metabolic activation by S9 fraction of rat liver. Hence, the specific role of the compound in vivo remains uncertain because the knowledge about direct exposure through areca nut is still unknown.

Involvement of enzymes in the metabolism of arecoline and arecoline N-oxide
A study of arecoline metabolism along with metabolism of its metabolites arecaidine and arecoline N-oxide has revealed basic routes that these compounds undergo. It involves de-esterification, N-oxidation and reduction of the double bond leading to the formation of metabolites, including mercapturic acids, mercapturic acid derivatives and nipecotic acid derivatives (Nery, 1971;Giri et al., 2006Giri et al., , 2007.

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According to Patterson and Kosh (1993), cytochrome P450 (CYP450) family of enzymes do not play a significant role in arecoline metabolism because most of arecoline was metabolized by mice liver homogenate even in the presence of nonspecific CYP450 inhibitor. However, these enzymes might be involved in the metabolic activation of N-nitrosamine compounds formed from arecoline. This is supported by a study conducted in a bacterial test system where metabolic activation of MNPN, MNPA and NGL was observed to be carried out by human CYP450 enzymes, especially by members of family CYP2A and CYP1A1 (Miyazaki et al., 2005). CYP enzymes activate N-nitrosamines and the products formed, thereby lead to alkylation of nucleic acid base pairs (Miyazaki et al., 2005). Genetic polymorphisms of CYP enzyme encoding genes (CYP2A6 and CYP1A1) have been found to be associated with oral cancer (Kao et al., 2002;Topcu et al., 2002).
In contrast to CYP450, flavin-containing monooxygenases participate in the metabolism of arecoline to its N-oxide (Giri et al., 2007). No association study has been found so far between the FMO polymorphism and oral cancer. As arecoline N-oxide is biologically active and might have a prominent role in areca nut mediated fibrotic disorders, the existence of polymorphic variants of the gene encoding this enzyme in the general population might develop predisposition towards development of areca nut mediated fibrosis. FMO-1 carries out the process most efficiently of all the other isozymes (Giri et al., 2007). It is abundantly expressed in the kidney (Zhang and Cashman, 2006). An association has been found between betel quid chewing and chronic kidney disorder in a population-based study. However, the association was influenced by several other factors (Hsu et al., 2011). In addition, a portion of the formed N-oxide undergoes reduction and forms the parent compound arecoline. CYP450 family of enzymes plays a role in this type of deoxygenation (Krueger and Williams, 2005;Montellano, 2013).
Carboxylesterases are involved in the metabolism of arecoline to arecaidine (Patterson and Kosh, 1993). In vitro, mercapturic acid formation from arecoline does not require any enzymatic assistance (Boyland and Nery, 1969). Therefore, under in vivo condition, mercapturic acid formation from the parent compound and metabolites might involve reaction with glutathione without the involvement of any enzymes. Although glutathione S-transferases (GSTs) participate in the phase 2 metabolism of xenobiotics, producing mercapturic acids (Hayakawa, 1977), the involvement of GSTs in arecoline metabolism is not known.
Apart from the study conducted by Patterson and Kosh (1993) and Giri et al. (2007), direct involvement of enzymes in the metabolism of arecoline in mammals has not been studied. In a study carried out by Chiang et al. (2007), arecoline has been found to suppress the expression of several phase I and phase II xenobiotic-metabolizing enzymes, which might indirectly affect the metabolism of arecoline itself. Moreover, mechanism of formation of other metabolites like nipecotic acid derivatives and the aldehyde derivative of arecoline (1-methyl-3,4-dehydropiperidine-3-carboxaldehyde) has not been studied yet.  (Shirname et al., 1983). Induced sister chromatid exchange in mice bone marrow cells (Panigrahi and Rao, 1984).
Weakly induced DNA single strand break in cultured human epithelial cells (Sundqvist et al., 1989). Induced both collagen formation by fibroblasts and collagen phagocytic ability reduction of fibroblasts (Harvey et al., 1986;Tsai et al., 1999). Had no effect on the myofibroblastic transdifferentiation of fibroblasts (Chang et al., 2013b). Induced senescence, DNA double stranded breaks along with transforming growth factor-β and matrix metalloproteinase-2 expressions in fibroblasts (Rehman et al., 2016).
Possibly harmful

3-methylnitrosaminopropionitrile
Induced tumour in several organ of exposed rats (Wenke et al., 1984b). Induced modification of DNA base pair in exposed rats Rivenson et al., 1988). Weak inducer of DNA single strand breaks in cultured human epithelial cells (Sundqvist et al., 1989).
Weakly mutagenic in bacterial tester strains (TA100, TA 98) (Wang and Peng, 1996). CYP450 and FMO mediated metabolism of arecoline N-oxide has been found to take place in the mitochondria of liver cells in a study conducted by Wang et al. (2018). Mitochondrial metabolism of arecoline N-oxide by the above-mentioned enzymes might be responsible for the generation of ROS, which mediates the toxic effects of the compound.

Conclusion
Apart from arecaidine and arecoline N-oxide, the biological effects of other metabolites also need to be elucidated. Some of them might be promptly modified by enzymes in a manner similar to mercapturic acids so that their excretion is facilitated, and subsequently, they pose a lesser threat for carcinogenic activity. Other compounds as well as metabolites that possess a high toxic potential might also be present in areca nut and oral cells might be directly exposed to them on the consumption of the nut. For example, both arecaidine and N-methylnipecotic acid are also present in areca nut (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2004;Hu et al., 2010). But as the toxicity of N-methylnipecotic acid is not known, its effect on the site of exposure cannot be determined.
Knowledge about the enzymes and genes that encodes them can provide an important insight into the metabolism of these xenobiotics. This will open another field of research correlating the differential expression and polymorphisms of these genes to an individual predisposition to oral cancer in betel nut consumers. Even the metabolites might be more potent in causing hazardous effects than the parent compound, as seen in the case of arecoline N-oxide.