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Genetically determined metabolism of nicotine and its clinical significance

1Department of Pharmacology, School of Medicine with the Division of Dentistry in Zabrze, Medical University of Silesia, Katowice, Poland; 2Department of Nuclear Medicine and Endocrine Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland; 3Silesian Medical College in Katowice, Mickiewicza 29, Katowice 40-085, Poland; 4Department of Pharmaceutics, School of Pharmacy, Virginia Commonwealth University and Affiliated with Center for the Study of Tobacco Products, Virginia Commonwealth University, Richmond, VA, USA

I of xenobiotic metabolism, as well as in the metabolism of certain endogenous substances, such as steroid hormones. The catalytic activity of CYP 450 includes a number of different reactions. These are mainly oxidations (C-, N-and S-oxidations) and dealkylations (O-, S-and N-dealkylations). The core element of the cytochrome activity is the electron transport chain in which crucial role plays the human NADPH-cytochrome P-450 oxidoreductase (Purnapatre et al., 2008;Di et al., 2009). The primary structure of the enzyme protein determines different isoenzymes of CYP 450. These isoforms of the enzyme have been classified into families and subfamilies. In human body, CYP1, CYP2, and CYP3 are involved in drug metabolism and biotransformation of xenobiotics (Purnaparte et al., 2008).
There are three CYP2A genes in humans (CY-P2A6, CYP2A7, and CYP2A13) and one pseudogene (CYP2A18). CYP2A6 expression is mainly found in the liver, but its protein or mRNA is also expressed in other tissues (Godoy et al., 2002;Ding & Kaminsky, 2003). CYP2A6 is responsible for the critical step in conversion of nicotine to cotinine in human liver. CYP2A6 is also responsible for further conversion of cotinine to trans-3'-hydroxycotinine (Nakajima et al., 1996). Different metabolic groups have been characterized due to genetic differences in the speed of metabolism, including poor-(PM), extensive-(EM) and ultra-rapid metabolizers (UM). The activity of CYP2A6 enzyme may be altered by genetic and environmental factors. Modification of CYP2A6 activity by drugs is a very important issue in medical and pharmaceutical practice (Di et al., 2009). The majority of cytochrome P-450 enzymes is present in the liver, mostly in the endoplasmic reticulum of hepatocytes. However, in much smaller amounts, they are also present in cells of other organs, such as the brain, kidneys, lungs, heart, and even skin.
The amount of CYPs in the brain parallels approximately 0.5-2% of that in the liver and is too low to significantly influence the overall pharmacokinetics of drugs and hormones in the body, but can play a role in modulating sensitivity to exogenous and endogenous compounds. Distribution of CYPs in the human brain varies among different brain regions. Within cortex, CYP2B6 is highly expressed in astrocytes surrounding cerebral blood vessels, whereas CYP2D6 is highly found in pyramidal neurons and also in the white matter. There are also differences between smokers and non-smokers. In the cerebellum of non-smokers, CYP2B6 and CYP2D6 are expressed in neurons within the molecular and granular layers, but are undetectable in Purkinje cells, however, in smokers, CYP2B6 and CYP2D6 are highly expressed in the Purkinje cells of the cerebellum Howard et al., 2003).
Smokers and alcoholics have higher levels of CYP2B6, CYP2E1, and CYP2D6 in specific brain regions. They may respond differently to certain drugs and toxins due to the elevated levels of CYPs in the brain. CYP2B6, which can also metabolize nicotine, is expressed in the brain. The CYP2B6 gene is polymorphic and smokers who are CYP2B6 slow metabolizers have increased withdrawal symptoms, higher craving and lower quitting rates Lerman et al., 2002).

METABOLISM OF NICOTINE
Nicotine is metabolized in the liver to six major metabolites (Fig. 1). The major metabolite is cotinine, to which about 70-80% of the absorbed nicotine is metabolized. This process consists of two stages. The first step is the formation of nicotine-Δ1'(5')-iminium ion, which is in balance with the 5'-hydroxynicotine. This step is catalyzed by CYP2A6 isoform. The second step involves oxidation of nicotine-Δ1' (5')-iminium ion to cotinine under the influence of cytoplasmic aldehyde oxidase . A small amount of nicotine (4-7%) is metabolized by flavin-containing monooxygenase 3 (FMO3) to another significant metabolite, nicotine N'-oxide. The nicotine N'-oxide does not undergo significant changes, except reduction to nicotine in the intestinal epithelium. In addition to oxidation reactions, nicotine is metabolized by glucuronidation to form Nbeta-(S) nicotine glucuronide. The last reaction is catalyzed by glucuronyltransferases (UGT). 3-5% of nicotine is converted in this reaction. A final important metabolite directly formed from nicotine through oxidative N-demethylation is nornicotine (Hukkanen et al., 2005). As much as 70-80% of nicotine is metabolized to cotinine in humans, but only 10-15% is excreted as free cotinine in urine. The major metabolites of cotinine are cotinine glucuronide and trans-3'-hydroxycotinine (Crawford et al., 1998;Raunio et al., 2001;Benowitz et al., 2009). Also, other CYP 450 isoforms, such as CYP2B6, CYP2D6, and CYP2E1, are involved in the metabolism 1, increase in pulse rate and blood pressure; 2, increase in plasma free fatty acids and the level of catecholamines in the blood; 3, desensitization of the GABAergic neurons by chronic stimulation by nicotine (addiction); 4, insulin resistance and predisposition to metabolic syndrome; 5, activation of signal transduction pathways, allowing the survival of damaged epithelial cells; 6, precursor of tobacco specific nitrosamines (TSNAs) through nitrosation; 7, substrate for arachidonic acid metabolites which cause increased cell division; 8, acceleration of tumor growth by increased angiogenesis mediated by β-adrenergic activation; 9, activation of nuclear factor kappa B (NF-kB)dependent survival of cancer cell and proliferation; 10, promotion of the growth of lung tumor by inhibiting anti-apoptotic pathway; 11, cause of resistance to the chemotherapeutic agents; 12, plays a contributory role in tobacco dependence; 13, modulation of the nAChR sensitivity to agonists. of nicotine (Hukkanen et al., 2005). Both, in vitro and in vivo studies indicate that CYP2A6 is mainly responsible for oxidation of nicotine to cotinine. In studies on the activity of microsomal enzymes, cotinine formation from nicotine and trans-3'-hydroxycotinine formation from cotinine, were correlated with the activity of coumarin 7-hydroxylase, which was strictly related to the activity of CYP2A6 (Cashman et al., 1992;Berkman et al., 1995;Nakajima et al., 1996). The ratio of the concentrations of 3-hydroxycotinine to cotinine in plasma and saliva is known as the nicotine metabolite ratio (NMR). This ratio is widely used for phenotyping CYP2A6 metabolic activity thanks to long half-life of cotinine (Dempsey et al., 2004). This non-invasive method, enabling to assess CYP2A6 activity, may be helpful in predicting the nicotine intake (Ross et al., 2016) and the effectiveness of smoking cessation therapies, like NRT (Chen et al., 2013).

DRUGS AFFECTING CYP2A6 ACTIVITY
CYP2A6 activity can be modified by drugs. There are several inhibitors, as well as inducers, of CYP2A6 among medicaments. It has been shown that 8-methoxypsoralen (a furanocoumarin used in the treatment of psoriasis, vitiligo and cutaneous T-cell lymphoma) is a potent, irreversible CYP2A6 inhibitor in vitro (Koenigs et al., 1997) and moderately effective human CYP2A6 inhibitor in vivo (Kharasch et al., 2000). Pilocarpine, a natural cholinomimetic alkaloid derived from Pilocarpus jaborandi, inhibits CYP2A6 and other enzymes involved in drug metabolism in mice and humans (Kimonen et al., 1995). Tranylcypromine, a nonselective monoaminooxidase inhibitor for the treatment of depression, is a selective potent CYP2A6 inhibitor (Taavitsainen et al., 2001). Also, selegiline, a selective monoaminooxidase B inhibitor used in the treatment of Parkinson's disease, was shown to be a CYP2A6 inhibitor (Siu & Tyndale, 2008). Antifungal drugs, such as klotrimazole, ketoconazole, and mikonazole are strong inhibitors of the CYP2A6 activity (Draper et al., 1997). Dexamethasone induces CYP2A6 activity by facilitating HNF4α (hepatic nuclear factor 4α) binding to HNF4α-RE (HNF4α response element) which increases the CYP2A6 transcriptional activity. This may be caused by changed histone H4 acetylation in proximal promoter of the CYP2A6 gene (Onica et al., 2008). Rifampin and phenobarbital also belong to CYP2A6 inducers (Mau-rice at al., 1991;Rae et al., 2001). Interestingly, atorvastatin, fluvastatin, and rosuvastatin are associated with activation of pregnane X receptors (PXR) and increase in the CYP2A6 activity. A degree of this effect depends on statin and its enantiomer (Korhonova et al., 2015).

GENETIC POLYMORPHISM OF CYP2A6
There are more than 37 different allelic variants of CYP2A6 (Gaedigk et al., 2018). Studies have shown that among homozygous individuals with the CYP2A6 gene, there is a lack of detectable cotinine level in the plasma (Nakajima et al., 2000;Swan et al., 2009). Wild type refers to CYP2A6* 1A. Polymorphic variants of this enzyme's gene may cause alterations in enzymatic activity of the encoded enzyme. Each allele accompanied with another allele can form a homozygote or heterozygote of the CYP2A6 locus. CYP2A6*2 goes together with CYP2A6*2 composing homozygote with slow metabolism. Similarly, CYP2A6*2 and CYP2A6*4 form a "poor metabolizer" (PM) heterozygote and both ) also form a PM heterozygote when present together with CYP2A6*1 . CYP2A2*1 determines rapid metabolism of nicotine, whereas CYP2A6 alleles *2 and *4 are responsible for the reduced metabolism ratio. Their occurrence is closely associated with the ethnicity. Alleles *2 and *4 are present in 1% of Caucasian and 20% of Japanese populations, respectively . In vivo and in vitro studies on the CYP2A6*2 variant had shown that this allele determines the total lack of activity of the encoded enzyme (Fernandez-Salguero et al., 1995). CYP2A6*4 with total deletion of the gene has no enzymatic activity (Oscarson et al., 1999). Variants associated with a single nucleotide polymorphism (SNP) are: CYP2A6*2, CYP2A6*5, *6 CYP2A6 , CYP2A6, CY-P2A6 *7 and *9 (O'Loughlin et al., 2004). CYP2A6*10 has reduced enzymatic activity (Yoshida et al., 2002). The same polymorphism could have a different effect on different substrates, thus it should be examined in regard to the individual substrates (Tyndale & Sellers, 2002). The differences also apply to medicines used in cancer treatment, such as letrozole. CYP2A6*12 heterozygote individuals are intermediate nicotine metabolizers and simultaneously slow letrozole metabolizers (Fukami et al., 2005).

POTENTIAL ROLE OF CYTOCHROME P-450 POLYMORPHISM IN CARCINOGENESIS
The ability of CYP2A6 enzymes to activate certain procarcinogens present in the cigarette smoke makes their polymorphism more significant. The complexity of carcinogens and compounds present in tobacco smoke results in an indirect link between a particular carcinogen and specific tumor (Rossini et al., 2008).
Hecht and others (Hecht et al., 2000) reported a new pathway of 2'-hydroxylation of nicotine via 2'-hydroxynicotine, which leads to the formation of 4-(methylamino)-1-(3-pyridyl)-1-butanone. This compound can be converted in vitro into a carcinogenic nitrosamine. However, endogenous production of NNK in the human body has not yet been established. Taking into consideration the ability of CYP2A6 metabolism to generate nitrosamines, London and others (London et al., 1999) hypothesized that individuals with slower metabolism may have a lower risk of smoking-associated lung cancer. Lower rate of nicotine metabolism by CYP 450 enzymes is correlated with a lower risk of addiction to nicotine. According to Tyndale and others (Tyndale et al., 2002), individuals with alleles responsible for defective nicotine metabolism had a decreased risk of becoming a smoker or they smoked less in comparison to extensive nicotine metabolizers. Slower metabolizers may also activate less tobaccorelated procarcinogens in comparison to extensive metabolizers. The type of metabolism also affects nicotine obtained from NRT. Also, the CYP2B6 polymorphism has influence on the risk of lung cancer development by affecting enzymatic activation of tobacco -specific nitrosamines, both, in conjunction with and regardless of the activity of the CYP2A6 enzyme (Wassenaar et al., 2013).

IMPLICATIONS FOR SMOKING CESSATION THERAPY
Smokers that have alleles associated with high activity of the enzyme (CYP2A6*1/*1B) experience serious symptoms of abstinence during breaking the habit. This correlation is more visible in NRT-using individuals when the delivered dose of nicotine is reduced with the duration of therapy (Kubota et al., 2006). People with a low rate of nicotine metabolism smoke less cigarettes and therefore need lower doses of nicotine from NRT (O'Loughlin et al., 2004). Individuals with normal enzyme activity become more easily addicted than those with a lower rate of metabolism and expressing CYP2A6*9, CYP2A6*2 or CYP2A6*4 alleles (Audrain-McGovern et al., 2007). According to Lerman and others (Lerman et al., 2006), in the group using transdermal nicotine, the intensity of cravings increased linearly with the increased NMR. It has been also shown that drugs that inhibit the function of the CYP2A6 reduce the number of cigarettes smoked per day . Methoxsalen  (Gu et al., 2000) CYP2A6 CYP2A6*7 CYP2A6*4 CYP2A6*10 poor the impairment of the enzyme is associated with protection from hepatoxic effect of drugs, such as valproic acid (Sadeque et al., 1997) CYP2A6 CYP2A6*3 poor associated with protective role against nicotine addiction (Oscarson et al., 1999) CYP2A6 CYP2A6*1 extensive wild type of gene is associated with higher risk of lung cancer, as it was proved to be more frequent in cancer patients than in control group (Miyamoto et al., 1999) CYP2A6 CYP2A6*4C poor unable to activate nicotine-derived carcinogens, N-nitrosamines such as NNK, which results in lower risk of cancer (Miyamoto et al., 1999) CYP2A6 CYP2A6*1B ultrarapid higher NMR in comparison to wildtype homozygotes. Association with smoking behaviour and response to NRT. Increased likelihood of being a smoker and higher amount of cigarettes smoked per day (Gambier et al., 2005) CYP2A6 CYP2A6*7 poor lower cigarette consumption per day, shorter smoking duration (Liu et al., 2011) CYP2A6 CYP2A6*17 poor increased ability to quit smoking habit (Ho et al., 2009) CYP2A6 CYP2A6*35 poor increased ability to quit smoking habit (Al  is an example of CYP2A6 inhibitor (Damaj et al., 2007). In pharmacokinetic studies on methoxalen, after its oral administration post an overnight period of abstinence, higher concentrations of nicotine in the plasma were observed. A reduced desire for smoking was also observed . Therefore, it is thought that inhibition of the CYP2A6 activity could be considered as a smoking cessation strategy. The polymorphism of CYP2A6 is also connected with the age of smoking initiation and the age of occurrence of chronic obstructive pulmonary disease (COPD) in smoking patients (Siedlinski et al., 2011). Inhibition of CYP2A6 could enhance the effectiveness of nicotine gums, patches and other forms of NRT, due the fact that these forms provide about 50% of the plasma concentration obtained by smoking (Sellers et al., 2003;Benowitz et al., 1997).

CLINICAL IMPLICATIONS OF NICOTINE METABOLISM
Metabolism of nicotine, mainly through CYP2A6, also has many clinical implications, including efficacy and safety of NRT and occurrence of several diseases. This makes the type of nicotine metabolism a potential predictor of clinical outcomes in patients with cardiovascular disease, addicted to nicotine, and those using NRT, as well as taking other medications. The most significant clinical implications related to the type of nicotine metabolism are summarized in Table 1.

CONCLUSIONS
The variety of cytochrome P-450 isoforms that take part in nicotine metabolism makes this topic significant due to its clinical consequences. In reference to CYP2A6, likelihood of becoming a smoker, consumption of cigarettes per day, and response to nicotine replacement therapy are often mentioned in contrast to other processes, such as metabolism of drugs and carcinogenesis. Genotyping toward CYP2A6 may be a helpful clinical tool in a group of patients using nicotine, irrespectively of the way of its administration, in order to assess their type of nicotine metabolism and its clinical significance.

Conflicts of interest
LK works as an expert for the Polish National Committee for Standardization and for the European Committee for standardization of requirements and test methods for e-liquids and emissions. LK was also an employee of the Institute of Occupational Medicine and Environmental Health. One of the institute's objectives is outsourcing for the industrial sector, including manufacturers of e-cigarettes. However, this has no influence on the study's design.