Cigarette Smoking Reduces BDNF in Heavy Smokers


 Background: Cigarette smoke is a powerful environmental modifier of DNA methylation. BDNF is a neurotrophin family of growth factors and is important in growth, progression, regeneration, survival, maintenance, and function of neurons. In this study, we aimed to evaluate the epigenetic changes, serum level, and gene expression of BDNF, in cigarette smokers. Methods: To assess the BDNF DNA methylation, peripheral blood samples were obtained from 237 cigarette smokers and 90 healthy nonsmokers as controls. DNA methylation was evaluated with MS-PCR. Gene expression and serum level were carried out with qRT-PCR and ELISA assay respectively. Results: The results from MS-PCR showed that DNA methylation of BDNF gene is more significant in heavy smokers than healthy nonsmokers as control (p-value <0.05). Serum level of BDNF and gene expression is significantly lower in heavy smokers than in healthy nonsmokers (p-value <0.05). Conclusion: In conclusion, heavy smoking can modify BDNF gene epigenetics. The serum levels of BDNF and the gene expression decreases in heavy smokers compared to healthy nonsmokers.


Background:
Smoking is now considered as a preventable cause of death in the United States. There are 480,000 deaths related to smoking each year, according to the Centers for Disease Control. 8.6 millions of people have at least one serious illness due to smoking. Cigarette smoking is a key risk factor for coronary heart disease and the primary factor in lung cancer [1]. Smoking is a powerful environmental modi er of DNA methylation [2]. Brain-derived neurotrophic factor (BDNF) is important for the maintenance, survival, and regeneration of neurons in the adult brain. BDNF, is a member of the neurotrophin family of growth factors that was discovered in 1982. The BDNF gene in human and rat is located on chromosomes (1q14.1) and (2q3) and has 11 exons and 9 exons, respectively [3,4]. There are two types of BDNF in the human body: pro-BDNF and mature BDNF. The pro-BDNF 32 KD consists of 247 amino acids that break in the cells and form the mature BDNF that secrets from the neurons [5][6][7][8]. BDNF is strongly expressed in the central and peripheral nervous systems. It is one of the most abundant neurotrophins in the brain with high concentrations in the cortex and the hippocampus. BDNF has a role in regulating the secretion of neurotransmitters with a key effect on serotonergic, dopaminergic, glutamatergic, and plasticity mechanisms such as long-term potential and mechanisms in learning and memory [9]. Many studies have shown a close link between BDNF and some diseases like schizophrenia, Alzheimer's disease, mood disorders, and Parkinson's disease [10][11][12][13][14]. Some studies have shown that BDNF probably has a necessary role in neuroimmune regulation of mood disorders. The immune cells and immune factors like cytokines play an essential role in the regulation of BDNF; for example, interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) affect the behavior through direct functions in the nervous system. These functions activate in ammatory signal pathways and reduce nerve growth factors like BDNF. On the other hand, the regulation of the BDNF-mediated signaling pathways is required for the process of the immunomodulatory. The mechanism that how BDNF speci cally regulates the neuroimmune axis in mood disorders is unclear [15,16]. Smith PH, et al. found a high prevalence of smoking among patients with depression, general anxiety disorders, and mental disorders. They found that smoking is more common among individuals with mood, anxiety, and substance use disorders, with the highest rates among those with bipolar and substance use disorders [17]. To note the important function of BDNF in the nervous system, we decided to evaluate the epigenetic changes of the BDNF gene in the smokers' individuals. Also, we compared the serum level of BDNF and the expression of the mRNA, between smokers and nonsmokers' subjects.

Methods:
Blood samples were obtained from 237 cigarette smoker individuals (mean age = 34 ± 12) and 90 healthy nonsmokers as controls (mean age = 36 ± 11), with no history of psychiatric illnesses. Before data collection, all respondents were informed about the aims of the study and data con dentiality and gave written informed consent. All participants took part in the survey voluntarily. Exclusion criteria were as follow: a history of primary neurologic diseases such as Parkinson's disease or depressive disorders, schizophrenia, suicide disorder, Alzheimer's and Huntington disease, alcohol usage. Smoker individuals were classi ed into three groups according to the Fagerstrom protocol: sever, moderate and low users.

Bisul te Modi cation Of Genomic Dna And Methylation Analysis:
Genomic DNA was extracted from whole blood leukocytes according to the protocol described with the (Pars Tous Company, Iran) kit. Brie y extracted DNA samples were treated with sodium bisul te and then PCR ampli ed using primers speci c for either the methylated and modi ed unmethylated promoter region of each growth factor genes. The primers are listed in Table 1. In all MSP reactions, DNA from normal leukocytes and universal human methylated DNA standards from Zymo Research (ZYMO Research, Freiburg, Germany) were used as unmethylated (negative) and methylated (positive) controls, respectively.

Methylation Speci c Pcr (ms-pcr):
The results from MS-PCR showed that the percentage of the people that their BDNF gene ( in exon I, in promoter I (was methylated, was signi cantly higher in heavy smokers than in healthy nonsmokers, Table  4, (P-value = 0.001). Also, the rate of cigarette dependence directly correlates with the methylation status in the subjects. (Fig. 1). Figure 1. Correlation of nicotine dependence with BDNF gene methylation status. the percentage of the people that their BDNF gene was methylated, was higher in the heavy smokers > medium smokers > low smokers. ** P-value < 0.001 Quantitative Real-time Polymerase Chain Reaction: The RT-PCR products were quanti ed using 2 −∆∆ct equation. The threshold cycle of BDNF gene was used to calculate the relative expression of the gene in smokers and control groups. The results showed that the relative expression of BDNF gene in heavy smokers is signi cantly (p-value < 0.001) lower than the healthy nonsmokers (Fig. 2). Enzyme-linked Immunosorbent Assay (elisa): The serum level of BDNF was signi cantly (p-value < 0.05) lower in heavy smokers than healthy nonsmokers; these data con rm the results from real-time PCR (Fig. 3). 237 cigarette smoker individuals and 90 healthy nonsmokers as controls participated in this study. All participants were men, unfortunately, women were not willing to participate due to social and cultural reasons. According to the Fagerstrom standard protocol, people were divided into three groups as shown in table 3. In summary, the basis of the scale for nicotine dependence is the number of cigarette consumption per day, and a questionnaire that evaluates the level of nicotine dependency in the individual.

Methylation speci c PCR (MS-PCR):
The results from MS-PCR showed that the percentage of the people that their BDNF gene ( in exon I, in promoter I (was methylated, was signi cantly higher in heavy smokers than in healthy nonsmokers, table 4, (P-value =0.001). Also, the rate of cigarette dependence directly correlates with the methylation status in the subjects. (Fig.1).

Fig1
. Correlation of nicotine dependence with BDNF gene methylation status. the percentage of the people that their BDNF gene was methylated, was higher in the heavy smokers >medium smokers> low smokers. ⃰ ⃰ P-value <0.001 Quantitative real-time polymerase chain reaction: The RT-PCR products were quanti ed using 2 -∆∆ct equation. The threshold cycle of BDNF gene was used to calculate the relative expression of the gene in smokers and control groups. The results showed that the relative expression of BDNF gene in heavy smokers is signi cantly (p-value <0.001) lower than the healthy nonsmokers (Fig.2). The serum level of BDNF was signi cantly (p-value <0.05) lower in heavy smokers than healthy nonsmokers; these data con rm the results from real-time PCR (Fig.3).

Fig3. Comparison between heavy smokers and healthy nonsmokers in the serum level of BDNF.
The results from the groups of moderate and low smokers did not show any signi cant difference, data not shown. ⃰ ⃰ P-value <0.05 Discussion: In this study, we aimed to evaluate nerve growth factor BDNF gene methylation in smokers. Also, we compared the serum level of BDNF and its gene expression between cigarette smokers and healthy nonsmokers. Our results showed that BDNF gene methylation was signi cantly higher in heavy smokers than healthy nonsmokers. Data from real-time PCR and ELISA assay con rm the results from MS-PCR. BDNF gene expression and serum concentration were signi cantly lower in heavy smokers than healthy nonsmokers. These data showed that BDNF gene methylation correlated with lower expression of the gene and serum level of the protein. Although serum level of BDNF, mostly originated from the brain, BDNF is stored in the platelets in a signi cant amount and is released when it is necessary [18]. Our ndings are consistent with the previous study by M. Yeom et al. which showed that repeated exposure to the nicotine reduces the BDNF expression and protein level [19]. The lower BDNF level may correlate to the reduction of the bene cial effects of this growth factor. A few studies investigated nerve growth factor BDNF and epigenetics changes in smoker's healthy people. Toledo-Rodriquez et al. found that adult individuals who had been exposed to prenatal smoking showed DNA methylation of the BDNF gene, this strongly leads to long-term downregulation of BDNF expression [20]. In another study, the decrease in BDNF mRNA and protein was contributed to the behavioral changes in male mice exposed to smoking. They suggested that epigenetic modi cation may underlie long-lasting changes in the plasticity and development of the brain. These changes may lead to laterneurodevelopmental problems and it may transfer to the next generation [21,22]. In a study, it has been shown that cigarette smog modi es DNA methylation of BDNF gene, and some other special genes like AXL, PTPRO, C11orf52, FRMD4A, CYP1A1, AhRR, FOXP3, TSLP, IGF2 in fetus, in smoking mothers against nonsmokers [23]. Zhang et al. showed that smoking severity is associated positively with serum BDNF levels, this contradiction may be due to the limited number of samples in their study and difference in the age of the participants, also while they measured fasting BDNF, we checked random BDNF in the serum, nicotine dose of injection may be another factor leading to different results [24]. Kenny et al. demonstrated, in an animal study, that acute nicotine administration signi cantly decreases the BDNF gene expression in the rat hippocampus [25]. Bhang et al. found that plasma concentration of BDNF is lower in smoker groups but its concentration increases after abstinence during 1-2 months [26]. All together more studies are needed to evaluate the BDNF exchange in the smokers speci cally. For the future studies, we suggest to compare BDNF level between the groups of active smokers, former smokers and non-smokers.

Conclusion
Heavy smoking can modify BDNF gene epigenetics and its serum level. To note the role of BDNF in nerve growth, regeneration, and maintenance, it may be an important issue to be considered in heavy smokers to prevent possible damages to the nervous system. Declarations Ethics approval and consent to participate: Prior to data collection, all respondents were informed about the aims of the study and data con dentiality, and gave written informed consent. All participants took part in the survey voluntarily.

Abbreviations
Consent for publication: not applicable.
Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Competing interests: The authors have declared that no con ict of interest exists ( nancial, non-nancial) and they have consented to the publication.
Funding: This study was funded by Fasa University of Medical Sciences, NO. 95123. Behnoosh Miladpour (who is funded) had several roles that are described at the "Authors' contributions" section.  Figure 1 Correlation of nicotine dependence with BDNF gene methylation status. the percentage of the people that their BDNF gene was methylated, was higher in the heavy smokers >medium smokers> low smokers. ⃰ ⃰ Pvalue <0.001 Comparison between heavy smokers and healthy nonsmokers in the serum level of BDNF. The results from the groups of moderate and low smokers did not show any signi cant difference, data not shown. ⃰ ⃰ P-value <0.05