Genetic variability in the neurobiology of nicotine dependence: effects on smoking behavior

Abstract Background Smoking dependence is a chronic disease and a public health problem. The neurobiology of nicotine addiction can explain smoking behavior. This system has genetic variability that has been associated with vulnerability to dependence. Genetic variability in the neurobiology of smoking can help to understand why individuals exposed to drugs may or may not become addicted. Objective This study aims to address genetic variability in the neurobiology of smoking addiction with a focus on polymorphic genes related to the nicotinic response and the dopaminergic reward pathway. Method This work involved a search of the main scientific research on genetic variability in the neurobiology of smoking and its effects on smoking behavior. One hundred and five studies were selected, most of which highlighted polymorphisms in the genes of nicotinic receptors, dopamine receptors, and nicotine metabolism. Results The majority of studies have focused on genes related to the activation of the dopaminergic reward system by nicotine. Combinations between different polymorphisms were also highlighted, showing that interactions can determine a genetic profile of predisposition to smoking addiction. Additionally, gender and ethnicity were identified as relevant factors. Conclusion Knowledge of the genetic bases involved in the individual response to smoking can enable a better understanding of inter-individual differences in smoking behavior, and contribute to improving the treatment of addiction.


INTRODUCTION
Tobacco smoking is a public health problem internationally recognized as a chemical dependency, with industrial cigarettes being considered the most important form of consumption 1,2 .According to the Tobacco Atlas, 5.7 trillion cigarettes were consumed worldwide in 2016 2 .Tobacco and tobacco smoke contains more than 8 thousand substances, among which nicotine, a psychoactive substance, is responsible for the addictive effects 3 .The verification of this psychoactive role means smoking is classified, according to the Review of the International Classification of Diseases and Related Health Problems (ICD10), in the group of mental and behavioral disorders related to the use of psychoactive substances 4 .
Tobacco smoke is the main cause of preventable mortality and morbidity in the world and accounts for the deaths of 8 million people per year.Of these, 1.2 million are non-smokers exposed to secondhand smoke 1 .For instance, in 2017, smoking was associated with 12.6% of the total deaths in Brazil 5 .According to data from Vigitel 2019, the total percentage of smokers aged 18 or over in Brazil was 9.8%, with 12.3% among men and 7.7% among women.Vigitel data points to a reduction in prevalence in both genders, although more pronounced in men 6 .This data indicates a new public health concern regarding the damage to women's health and an increase in tobacco-related diseases 1 .Although overall consumption has declined in recent years, the future path of global tobacco control is still uncertain and future projections are worrying.It is estimated that by the end of 2020, more than 10 million people will die from cardiovascular disease, chronic obstructive pulmonary disease, and lung cancer caused by tobacco use.Half of these deaths will occur during the productive years, with an individual loss of 10 to 20 years of life.In 2030, 80% of consumption-related deaths will occur in developing countries 7 .
Despite knowledge of these adverse health effects, smoking addiction explains why about 70% of smokers want to quit smoking, but have not succeeded.Of these, about a third are successful for just one day and less than 10% are abstinent for twelve months 8 , with cessation treatment being successful in only 35% of cases 9 .Smoking behavior is complex and multifactorial, determined by a combination of biological, psychological, and environmental factors 8 .Heredity is a strong component of tobacco use and its influence on dependence is at least 50% 10 .Genetic variations can influence up to 80% of characteristics of smoking behavior, such as initiation, persistence in smoking, and successful cessation 11 .
Genetic variability in the neurobiology of smoking and other addictions can help to understand why individuals exposed to drugs may or may not become addicted.In addition, knowledge of the genetic bases involved in the individual response to smoking can contribute to improving the treatment of addiction 12 .In this sense, this study aims to address genetic variability in the neurobiology of smoking addiction, focusing on polymorphic genes related to the nicotinic response and the dopaminergic reward pathway.

METHOD
This work involved a search of the main scientific research on genetic variability in the neurobiology of smoking and its effects on smoking behavior.Bibliographic searches were carried out between 2017 and 2020 in the PubMed, Scielo, and Medline databases.The following terms were used: genetic susceptibility, polymorphic genes, smoking, and nicotine addiction.Articles in Portuguese and English published between 2000 and 2020 were selected and qualified, according to their abstracts, as possible candidates to provide technical-scientific bases for this paper.At the end of the search, duplicate references and unavailable full studies were excluded.Two researchers analyzed and classified each abstract as being outside or within the scope.

RESULTS AND DISCUSSION
The search selected 105 articles that were used as a theoretical basis for the preparation of this work.Figure 1 presents the flowchart of the stages of the identification, selection, and inclusion of scientific articles.

Neurobiology of tobacco dependence
The neurobiology of smoking explains the molecular mechanism of the development of addiction based on the psychoactive character of nicotine.Inhaled nicotine is absorbed in the lungs from cigarette smoke and reaches the brain in 10 to 60 seconds, where it binds to nicotinic acetylcholine receptors (nAChR) in the mesolimbic system, producing the addictive effects of strengthening the smoking habit through activation of the dopaminergic reward system 13,14 .
In the presence of nicotine, the flow of dopamine increases in the mesolimbic system, activating brain circuits to regulate feelings of pleasure and reward.The mesolimbic dopaminergic system is the main neurobiological structure associated with addiction to smoking and plays a crucial role in reinforcement 15 .This system is mainly composed of the ventral tegmental area and the accumbens nucleus.These regions are related to the mechanisms of addiction to nicotine, such as craving, memory, emotions related to use, tolerance, and dysphoria due to abstinence.In addition to dopaminergic hyperactivity, serotonin is released in the acute phase of nicotine consumption.Additionally, prolonged exposure desensitizes the gamma-aminobutyric acid system (GABAergic), an inhibitor of brain systems, which reinforces the behavior of compulsive use of nicotine 13,14 .
The mechanism of activation of the dopaminergic reward system by nicotine occurs by binding nicotine to the nicotinic receptors of presynaptic neurons (nAChRs), thereby opening cationic channels and, consequently, causing neuronal depolarization.Under these circumstances, dopamine and other neurotransmitters are released in the synaptic cleft and bind to dopamine receptors (DRDs) in post-synaptic neurons, transmitting the signal between neurons.Dopamine is released from the synaptic neurons and some of it is captured by dopamine transporters (DAT) in the presynaptic neurons.After reuptake, dopamine can then be repackaged into vesicles for use in future neurotransmissions or can be degraded by monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) 14 (Figure 2).The genes involved in the neurobiology of smoking behavior have been investigated as candidates for individual susceptibility to smoking.Among these, special attention has been paid to those related to the nicotinic response and the neurotransmitter dopamine, considered the key to substance addiction and abuse 8,15 .
The first group of genes addressed in this study is directly related to the nicotinic response and is represented by the CYP2A6 metabolism gene (Cytochrome P4502A6) and by the genes encoding the nicotinic acetylcholine receptors, CHRN 8,16 .The second group includes the genes involved in the dopaminergic pathway, which are capable of interfering with the concentration of dopamine in the synaptic cleft.These are the dopaminergic receptor genes DRD2/ANKK1 and DRD4, the carrier gene of dopamine transporters (SLC6A3), and the genes of metabolism Dopamine B-hydroxylase (DBH), Catechol O-methyl transferase (COMT) and Monoamine oxidase (MAO) 13,15 .Table 1 presents the main characteristics of the genes studied in this work.

Polymorphism of CYP2A6
Approximately 80% of nicotine is converted into cotinine by the action of the enzyme expressed by the Cytochrome P4502A6 gene (CYP2A6).Thus, variations in this gene may alter the enzymatic activity, interfering with the concentration of nicotine that reaches the target sites 16 .Apparently, CYP2A6 functional polymorphisms, in addition to affecting smoking behavior, are also associated with an increased risk of lung cancer 17 .
An association between CYP2A6 genotypes and nicotine dependence has been reported using the Fagerström Test for Nicotine Dependence (FTND) to verify the degree of nicotine dependence.Carriers of the wild allele CYP2A6*1, called normal metabolizers, are the most susceptible to tobacco dependence due to needing to consume a greater number of cigarettes to maintain satisfactory levels of nicotine in the blood 18 .Other polymorphic variants of the CYP2A6 gene, such as the CYP2A6*9 and CYP2A6*12, have smaller enzymatic activity, and the CYP2A6*2 and CYP2A6*4 variants are associated with a total loss of activity.So, depending on the gene variant carried, an individual is categorized as a normal, intermediate, or slow metabolizer, with 100%, less than 75%, or less than 50% enzymatic efficiency, respectively 16,18 .Wassenaar et al. 18 observed a higher quantity of cigarettes consumed per day by individuals possessing normal metabolizer genes in comparison with those having slow metabolizers.Although other studies have not reported the same association 74 or even divergent results 75,76 , several works have shown a direct association between the CYP2A6 genotype of lower enzyme activity and the lower risk of becoming a smoker, less nicotine dependence, fewer cigarettes consumed and greater success in cessation [17][18][19][20][21] .

CHRN gene polymorphisms
The binding of nicotine to nicotinic acetylcholine receptors (nAChR) increases the concentration of dopamine and other neurotransmitters, promoting the activation of reward mechanisms, which is crucial for smoking behavior.Some variations found in the clusters of genes encoding nAChR in dopaminergic neurons are involved in the development of addiction.Special attention has been given to polymorphisms in the CHRNA4/CHRNB2, CHRNB3/CHRNA6, and CHRNA5/CHRNA3/CHRNB4 gene clusters 22,23 .
In 2007, an analysis of 3713 Single Nucleotides Polymorphisms (SNP) polymorphisms was published, highlighting the association of SNPs CHRNA3 (rs578776 C>T; rs1051730 C>T) and CHRNA5 (rs16969968 G>A) with smoking 21 .Based on this, several other studies have reported the association of gene variants of nicotinic acetylcholine receptors with nicotinic dependence in different groups, though very few have focused on the Brazilian population 24,77 .
Several authors have found associations between the variant alleles of rs578776, rs1051730, and rs16969968 with characteristics of smoking behavior, such as the risk for smoking, the number of cigarettes consumed, and the degree of dependence 17,22,[24][25][26][27][28]30,31 . TheseSNPs have also been associated with smoking cessation, but with inconsistent results.In general, studies have shown a significant association between the T allele of rs1051730 and the A allele of rs16969968 with a lower probability of cessation and, inversely, between the T allele of rs57877rs and a greater chance of cessation 29,[32][33][34] .But there are contradictory findings 19,77,78 .Genetic variations in CHRNB2 and CHRNA4 also seem to interfere with individuals' responses to drug treatments for smoking cessation.An example is the lower incidence of abstinence symptoms related to polymorphisms in CHRNB2 and CHRNA4 in individuals using the drug varenicline, which acts on neuronal nicotinic cholinergic receptors by stimulating the release of dopamine 35,40 .A recent study highlights the contribution of CHRNA4 (rs1044396 C> T) polymorphism in the choice of the best drug for anti-smoking treatment.According to this study, the effectiveness of varenicline is higher for patients with a CT or TT genotype than for those with CC 36 .
Swan et al. 40 showed an association of variant A of the CHRNB2 polymorphism (rs2072661 G> A) with nausea, an important adverse effect when discontinuing the use of varenicline 40 .Additional studies have reinforced the association of the variant T allele of rs1044396 (CHRNA4) and the wild G allele of rs2072661 (CHRNB2) with a greater possibility of quitting, lower risk of becoming a smoker, less dependence, and lower cotinine levels 19,35,[37][38][39][40][41][42] .

Polymorphisms of the DRD2 and DRD4 genes
Some functional variations have been found in the genes encoding dopamine receptors (DRDs) related to smoking.However, the most studied, for their association with smoking, are the polymorphs DRD2 rs1800497 and DRD4-VNTR 50,79 .Historically referred to as DRD2 Taq1A, the polymorph Taq1A (rs1800497 C> T) is a variation of the ANKK1 gene (Ankyrin Repeat And Kinase , where the presence of the A1 (T) allele is related to lower expression of the DRD2 dopamine receptor, which may interfere with the synaptic concentrations of the neurotransmitter.So, individuals with the A1 allele of this gene have a higher risk of being a smoker 80 , starting smoking at a lower age, have a higher degree of dependence, smoke more cigarettes, have shorter periods of abstinence, and make fewer attempts to quit smoking 30,43,44 .However, no association has been found in other studies 81,82 .Additionally, the A2 (C) allele may represent a risk in relation to the characteristics of smoking behavior 59,79 .
The SNP can also interfere with the response to pharmacological therapies for cessation.David et al. 45 found that the drug bupropion was effective only in smokers with the A2/A2 (CC) genotype 45 .Swan et al. 46 also observed that A2/A2 women were less likely to stop treatment with bupropion; however, the same associations were not observed in men 46 .An identical effect was also observed in the females for Nicotine Replacement Therapy (NRT) 47 .Other studies have reported an association between the A2 allele and a higher chance of abstinence and success in cessation 45,48,49 .However, this is not a unanimous result 59 .
A polymorphism of variable numerical repetition (VNTR) in the gene encoding the D4 receptor, DRD4, has also been investigated as a candidate for susceptibility to smoking addiction.Most studies have grouped the alleles into "long" (7 or more repetitions) or "short" (6 or less) 83 .Long alleles have been associated with lower expression of the gene in comparison with the short alleles 84 .The long allele of this polymorphism has been associated with an increased risk of smoking, greater cigarette consumption, a greater risk of initiation, and a greater degree of dependence [50][51][52][53] .The relationship between smoking cessation and these groups has also been studied by several authors, but with divergent results.Leventhal et al. 83 found that European individuals with the long allele group treated with bupropion have a greater chance of abstinence compared with a placebo group 83 .However, this result was not confirmed in other studies 85 .The influence of polymorphism was also studied for Nicotine Replacement Therapy (NRT) in individuals with European ancestors; this study showed that those possessing long alleles had a reduced probability of cessation 51 .Other studies have not confirmed the association between DRD4 VNTR and smoking behavior 86,87 .These differences in findings reinforce the complexity of nicotine addiction and the need for future studies.

Polymorphism of the SLC6A3 gene
The dopamine transporter (DAT), which is encoded by the SLC6A3 gene, mediates the active reuptake of dopamine from the synapse.Polymorphism of the SLC6A3 gene is linked to dopamine transport in the synaptic cleft.It is formed by the repetition of a 40-base pair sequence, which can interfere with the expression of the SLC6A3 gene that encodes the dopamine transport protein (D28).Alleles containing 10 and 9 repeats are the most frequent.The 10-repeats allele is associated with a higher rate of gene transcription and, therefore, with higher levels of the carrier protein 88 .Studies have shown that individuals with the 9-repeats allele are less likely to start smoking before the age of 16, have a shorter smoking time, longer periods of abstinence, and are more likely to quit smoking [54][55][56][57][58] .However, controversial results 89 and a lack of significant association 90 demonstrate the need for further studies on this subject.
A meta-analysis study showed that, although the genetic variations of SLC6A3 are related to dopamine regulation, there is a lack of evidence on their influence on smoking cessation, given the multifactorial nature of smoking 88 .However, this study reinforced the importance of gene interaction in susceptibility to smoking and showed that the interaction between the DRD2 Taq1A and SLC6A3 genes prolongs abstinence time and influences smoking cessation with the use of bupropion 55 .The results showed the role of gene-gene interaction in the probability of relapse: smokers possessing the A2 allele of DRD2 Taq1A and SLC6A3-9 had significantly higher rates of abstinence at the end of treatment and a longer latency period for relapse 55 .

Polymorphisms of the DBH gene
The DBH gene encodes the enzyme of the same name, which converts dopamine to norepinephrine; this means that lower levels of transcription or activity may result in higher concentrations of dopamine 51 .Several studies have reported functional polymorphisms in this gene related to smoking behavior 91,92 .The literature shows an association between rs77905 (A> G) polymorphism and nicotine dependence 59,60,92 .Johnstone et al. 59 reported an association between individuals with the GG genotype, in interaction with the A2/A2 genotype of the DRD2 Taq1A polymorphism, and greater persistence of smoking, as well as less effectiveness of cessation due to transdermal nicotine replacement 59 .However, McKinney et al. 92 observed that homozygous smokers of the G allele smoked fewer cigarettes than smokers with the A allele 92 .Some studies found no significant association between this SNP and smoking 93 .For instance, according to some authors, the SNP rs3025343 (G> A) is associated with smoking behavior, especially the G allele, which is related to smoking cessation 61,62 , but this is still controversial because other studies haven't confirmed this association 94 .

Polymorphism of the COMT gene
Some functional polymorphisms of the COMT gene involved in dopamine degradation linked to smoking have already been identified 63 .The rs4680 G>A variation (Val158/108Met) resulted in less enzyme activity.Therefore, the Val allele carriers showed a low level of the neurotransmitter dopamine and increased COMT activity in comparison with the Met allele 64 .
Several studies have shown an association between the Val allele and characteristics of smoking dependence, such as the risk of smoking initiation, a greater degree of dependence, and persistence 65,66 .Enoch et al. 63 , analyzing a sample of 342 individuals, observed this association in female smokers 63 .A similar result was found by Nedic et al. 64 in a study with 657 Caucasian men 64 .Additionally, an association between the Met/Met genotype and greater success in cessation has been reported 67 .A survey by Colilla et al. 95 , with a sample of 290 women, reported the success of NRT in smokers of Caucasian ethnicity with the homozygous genotype Met/Met in comparison with those with the Val/Val genotype.Another study of 749 Caucasians found that the Met/Met genotype is associated with higher abstinence rates 69 .However, some authors have reported different results [95][96][97][98][99] .

Polymorphisms of the MAOA and MAOB genes
Relevant variations for smoking in both monoamine oxidase genes, MAOA and MAOB, have been reported, since both are involved in the degradation of some neurotransmitters, such as dopamine 100,101 .As for the variability of the MAOA gene, research has focused on polymorphisms that affect smoking.One repetition polymorphism, the MAOA VNTR of the promoter region of the gene, which consists of 2 to 5 repetitions of a sequence of 30 base pairs, is related to smoking.Two alleles containing 3 and 4-repeats are most common 60,66,70,71,93,102,103 .The 4-repeats allele has been associated with a greater number of cigarettes consumed, compared to the 3-repeats allele, in Caucasian men with alcohol and tobacco dependence 71 .Similarly, the 4-repeats allele has been associated with higher FTND scores and a higher degree of dependence in women 70 .However, the data are not conclusive, since these findings have not been confirmed by other studies 66,93,103 .
Another polymorphism in the MAOA gene, called EcoRV rs1137070 1460C> T, is capable of altering the transcriptional activity of this gene 82,104 .In this case, the presence of the T variant reduces the risk of smoking, especially in Caucasians 72 , and in women 60 .However, some studies have found otherwise 66,102 .
Regarding variations in the MAOB gene, the A allele of the MAOB rs1799836 polymorphism (A> G) is associated with a lower risk of heavy smoking in men 72 .However, this association is contradicted by other studies 66,105 .Interactions between this SNP with other polymorphisms seem to interfere with the risk of smoking 70,73 .The association of the A allele with smoking risk was found only in association with the B12 genotype of a polymorphism known as the TaqIB of the DRD2 gene 73 .Other studies have shown that Japanese men with a combination of the MAOB rs1799836 G allele and the 3-repeats genotype of the VNTR MAOA started smoking later than those with other genotypic combinations 70

CONCLUSION
There are many genes involved in the neurobiology of smoking.Several are polymorphic and, admittedly, some of these variations can affect smoking behavior.
The majority of studies have focused on genes related to the activation of the dopaminergic reward system by nicotine present in cigarettes as candidates for susceptibility to addiction.Due to their association with a higher risk of smoking, the polymorphisms found in the genes CYP2A6, CHRNA3, CHRNA5, CHRNA4, CHRNB2, DRD2, DRD4, SLC6A3, DBH, COMT, MAOA, and MAOB were addressed.Among these, the SNPs CYP2A6 *1, CHRNA3 rs578776, CHRNA5 rs16969968, CHRNA4 rs1044396, CHRNB2 rs2072661, and DRD2 Taq1A seem to most influence the development of addiction and the worsening of specific characteristics of smoking behavior, such as the number of cigarettes consumed, the age of initiation, the efficiency of drug therapy and cessation.
The relevance of genotypic combinations between different polymorphisms reinforces that interactions between genes can determine a genetic profile of predisposition to addiction.In addition, the sex and ethnicity of the studied populations proved to be important factors in the investigations, especially in the context of a diverse and mixed population.The effects of genetic variability on smoking have received great attention.Advances in the field of pharmacogenetics have enabled a greater understanding of individuality in responses to drug therapies, both in terms of efficacy and adverse effects.Knowledge of the genetic variability of the neurobiology of smoking can help elucidate the issues inherent to smoking addiction and contribute to the development of more personalized and effective forms of treatment.However, the great variability of obtained results shows that this task is not simple.Apparently, it involves several factors.Therefore, more research is needed on this topic, especially considering population differences, the interference of environmental factors, and interactions between different polymorphisms.

Figure 1 .
Figure 1.Flowchart of the identification, selection, and inclusion of scientific articles