Volume 5, Issue 4 (Autumn 2019)                   Caspian J Neurol Sci 2019, 5(4): 168-174 | Back to browse issues page


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Mohammad Alizadeh M, Kazemi Nezhad S R, Babaei P, Keshavarz P. Comparing Oprm1 Gene Promoter Methylation in the Lymphocytes of Male Rats Addicted to Nicotine, Morphine, Methadone, and Buprenorphine. Caspian J Neurol Sci 2019; 5 (4) :168-174
URL: http://cjns.gums.ac.ir/article-1-294-en.html
1- Department of Genetics, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran
2- Neuroscience Research Center, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran
3- Cellular and Molecular Research Center, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran
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Introduction 
Drug addiction is one of the main concerns of health care organizations. According to the literature, there are around 29.5 million people (different reports from 15.3 to 43.1 million) in the world suffering from drug abuse. Biological, social, environmental, and genetic factors can influence the vulnerability to addiction [1, 2]. Studies on families, twins, and adopted children have shown that the odds of inheriting drug abuse disorders is moderate to high (0.30 to 0.70) [3]. However, genetic or environmental factors alone do not increase the tendency to addiction [4].
In drug addiction, compulsive drug-seeking and drug-taking behavior persist despite serious negative consequences. Addictive substances induce adaptive changes in the central nervous system and lead to tolerance, physical dependence, sensitization, craving, and relapse [5]. Morphine is one of the opioids which is widely used as a painkiller and an addictive drug [5-7]. Nicotine is the main ingredient of tobacco that causes addiction to smoke [7, 8]. Tobacco also is one of the leading causes of many diseases, mortality, and morbidity [9, 10]. Other opioids such as methadone and buprenorphine as an agonist of Mu-Opioid Receptor (MOR) are widely used for the treatment of dependence on other opioid drugs such as morphine [8].
Epigenetics refers to any change in the gene expression that does not alter the DNA sequence. DNA methylation and histone modification (methylation, Acetylation, phosphorylation, ubiquitylation, and sumoylation) are the most frequent epigenetic changes that regulate gene expression [11, 12]. Previous studies reported the involvement of DNA methylation and histone modifications in drug addiction [13-16]. Based on the reports, cytosine methylation induces epigenetic changes and transcriptional regulation of the mammalian genome. Cytosine methylation occurs on C5’ of the cytosine ring by DNA-methyltransferase in the C-G dinucleotides [17]. There are approximately 45000 CpG islands in the human genome and 19568 in the rat genome [18, 19]. Generally, the DNA methylation in promoter sites is related to reduction of gene expression [20, 21].
Opioid receptors are G protein-linked receptors activated through the interaction of downstream signaling with heterotrimeric G-protein [8]. Mu (µ), kappa (κ), and delta (δ) opioid receptors are respectively coded by three genes: OPRM1, OPRK1, and OPRD1 [22]. OPRM1 is a likely candidate gene for smoking and the main target in most drug therapies [1, 23]. Various studies have reported the higher level of DNA methylation in specific CpG sites in the Oprm1 promoter in addicted individuals to opioids compared to non-addicts [13, 22, 24, 25]. The μ opioid receptor is expressed in different cells, such as lymphocytes, macrophages, and neurons [26-28]. Hypermethylation of Oprm1 gene is followed by lower transcription of Oprm1 [29, 30]. Oprm1 gene has a binding site for several important translation factors such as Elongation Factor (EF2), CREB (cAMP response element-binding protein), and AP-1 (activator protein 1) [31].
Human studies on the OPRM1 gene of addicted people revealed more methylation rate of the promoter of this gene in the whole blood in addicted male compared with the control counterparts [22]. In Nielsen et al. studies on Caucasian and African-American populations, similar results were found in peripheral blood lymphocytes of the heroin addicts under methadone maintenance treatment (MMT) [13, 24]. Because addicted people usually use more than one drug, it is difficult to distinguish the exact effect of each drug on methylation in human, therefore this study was designed to evaluate the impact of four most commonly-used drugs (nicotine, morphine, methadone, and buprenorphine) on the methylation level of two CpG sites at the Oprm1 promoter in male Wistar rats separately. To the best of our knowledge, this is the first study to aim this critical goal.
Materials and Methods
Subjects
In this case-control study, 48 male Wistar rats with Mean±SD (200±30) g and aged two months were used. The animals were housed with free access to food and water in a 12 h light/dark cycle and at constant temperature of 22±2˚C. They were allowed to get habituated to the examiner and laboratory conditions (hence were handled at least three days before the experiments). All procedures concerning animal care were following the International Ethical Guidelines, the Declaration of Helsinki, and the Ethics Committee of Guilan University of Medical Sciences. 
Groups
The studied animals were classified into 6 groups with 8 rats in each group: 1. The control (intact) group; 2. the group receiving normal saline as a solvent for drug; 3. the group receiving nicotine: 0.4 mg/kg, SC injection for 5 days (N5260, Sigma-Aldrich, USA) [32]; 4. the group receiving incremental doses of morphine: 10 mg/kg on days 1-3 and 20 mg/kg on days 4-6 and 40 mg/kg, IP injection on days 7-9 (Daroupakhsh, Iran) [33]; 5. the group receiving methadone: 0.5 mg/kg, IP injection for 15 days (Daroupakhsh, Iran) [34]; and 6. the group receiving buprenorphine: 0.05 mg/kg, SC injection for 6 days (Faran, Iran) [35].
12 hours after the last injection, the male rats were anesthetized with ether. Their blood samples were taken from their venae cavae. To prevent the blood samples from clotting, we kept them in Ethylene Diamine Tetra acetic Acid (EDTA) tubes as an anticoagulant at -20˚C until DNA extraction. 
Genomic DNA extraction
DNA was extracted from blood lymphocytes of all rats using QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer’s protocol. Briefly, cells were lysed by optimized buffers and enzymes of this kit. DNA was adsorbed to the QIAamp membrane. Alcohol was added, and lysates loaded onto the QIAamp spin column. Wash buffers removed impurities, and pure, ready-to-use DNA was then eluted in water or low-salt buffer.
DNA methylation
The nucleotide sequence of the rat Oprm1 promoter region (chr1: 37533440-37536440, Rat.rn4) and CpG island sequence located at the promoter region of Oprm1, were downloaded from the UCSC Genome Browser website (genome.ucsc.edu). It shows that the Oprm1 gene is located at position 1p11; the 18 CpG sites are located in position 37535123-37535517. The primers were designed by the MethPrimer Web software (http://www.urogene.org/methprimer/) (Table 1). In the present research, 2 out of the 18 CpG sites (+33 CpG and -107 CpG) were investigated. As mentioned, the position of +33 CpG is an E2F transcription factor binding site. Also, the status of -107 CpG is binding sites of 2 transcription factors AP-1 and CREB [31].
To apply the MSP method, DNA is treated with sodium bisulfite, in which process, all unmethylated cytosine is converted to uracil [36, 37]. During the sulfonation reaction, however, methylated cytosine cannot be converted [38]. Therefore, the differences created by the reaction between the methylated and unmethylated cytosine can be distinguished by MSP. To characterize methylation percentage, genomic DNA (>300 ng) was treated with sodium bisulfite using the EpiTect Bisulfite Kit (QIAGEN) according to the manufacturer’s protocol. The final bisulfite-treated DNA was eluted in 20 μL elution buffer.
Methylation specific PCR
MSP is a PCR-based method for the analysis of methylation templates in CpG islands [36]. In the MSP method, 2 sets of methylated and unmethylated cytosine-specific primers of targeted sequences are used, and the PCR reaction is separately carried out. DNA amplification with primers was employed to amplify the Oprm1 promoter sites (+33 CpG and -107 CpG) (Figure 1). The final product size of PCR was 180 base pairs. Amplification was performed with 1.5 μL of bisulfite-treated DNA, 0.5 µL of each primers, 2.5 µL PCR buffer, 0.5 µL dNTPs, 0.75 µL MgCl2, 0.13 µL Taq DNA polymerase in 25 μL total reaction volume. The amplification was done over 5 min at 94°C; then 30 cycles, including 30 s at 94°C, 30 s at 59°C, 30 min at 72°C and a final 5-min elongation step at 72°C.
To evaluate the accuracy and quality of the desired fragments, we loaded them onto the 2% agarose gel electrophoresis. The place and status of PCR products in agarose gel were detected by staining with the fluorescent dye ethidium bromide under direct ultraviolet light. The results of electrophoresis in agarose gel were analyzed by observing the ultraviolet ray using gel documentation (Figure 1).
Results 
Gel electrophoresis of amplified DNA showed DNA bands in the row of PCR created by unmethylated primers. Figure 2 shows the results of the PCR process in which all samples have a positive response amplified by the unmethylated primer. Comparing the two CpG sites in the Oprm1 promoter in 48 rats in 6 groups, no change was observed at the methylation status of +33 and -107 CpG sites in Oprm1 promoter (Figure 2).
The Chi-squared analysis is a suitable test for the investigation of frequency differences between studied groups. The frequency of unmethylated cases in groups was less than 4. Therefore, the data did not apply to this analysis. Table 2 presents the frequency of the methylation status of the groups.
Discussion 
The results of the present study showed that nicotine, morphine, methadone, and buprenorphine in the doses used here led to the addiction of the male Wistar rats, demonstrating particular symptoms and behaviors, including ecstasy, solace, disquietude, seeking behaviors, standing, grooming, anxiety, and other abnormal signs such as constipation and diarrhea. These results are in agreement with the previous findings [39]. Surprisingly no change was observed in the methylation of Oprm1 gene promoter sites of blood lymphocytes. Our findings are contradictory to the previous studies carried out on the brain nucleus [31]. 
To the best of our knowledge, there is no study on methylation of Oprm1 gene promoter in lymphocytes of animals addicted to nicotine, morphine, methadone, or buprenorphine. However, human studies revealed the positive results of methylation of Oprm1 gene promoter in the blood (sits 13, 22, 24, and 25). In a recent study by Chorbov et al. on DNA methylation in 24 CpG sites of Oprm1 promoter in male opioid abusers (extracted from their blood samples), significant DNA methylation was found at 7 CpG sites (sites 5, 9, 10, 11, 18, 23, and 24). They further reported a high level of DNA methylation only in 2 CpG sites in the DNA extracted from the sperm samples of the addicted individuals [22].
Although we found negative results from the methylation in CpG sites of Oprm1 (-107 CpG and +33 CpG), methylation might have occurred in other CpG sites. Then, the absence of methylation in our study could be accompanied with an inactive transcription of the gene [40, 41].
In addition, the study of DNA methylation on peripheral blood lymphocytes of heroin addicts under MMT of the Caucasian population showed that methylation is significantly associated with the two CpG sites (18 and 84) in the Oprm1 gene promoter [13]. These observations were later replicated by Nielsen et al. in 2010. They reported that the level of DNA methylation in the Oprm1 gene promoter was higher in heroin addicts than in the control group in African-American and Hispanic populations. A comparison between these two populations revealed that the level of methylation in the Oprm1 gene promoter differs by geographical features [24].
Differences between human and animal studies might be related to the fact that addicted people often use a variety of drugs which can affect methylation rate cumulatively. The strong point of the present study relates to its capability to distinguish and specify the contribution of each drug to methylation. However, the limitation of our study was the evaluation of the methylation of other CpG regions or exons. Assessing all CpG sites in the promoter or exons might better explain the negative or positive epigenetic effect of nicotine, morphine, methadone, and buprenorphine.
One possible explanation for negative results on methylation of the Oprm1 gene promoter in male Wistar rats following addiction drug usage might be the insufficiency of either the doses of drugs or duration of treatment. Also, these drugs might not have been capable of methylating Oprm1 gene promoter in rats and probably cannot be considered as epigenetic substances in this species. Thus, these animals may have an unknown protective mechanism against epigenetic changes to addiction. 
Conclusion 
The current study shows that use of nicotine (0.4 mg/kg, SC injection for 5 days), morphine (10 mg/kg on days 1-3 and 20 mg/kg on days 4-6 and 40 mg/kg, IP injection on days 7-9), methadone (0.5 mg/kg, IP injection for 15 days) and buprenorphine (0.05 mg/kg, SC injection for 6 days) do not induce methylation of Oprm1 gene promoter sites in the blood samples of the male Wistar rats.
The epigenetic mechanism is triggered by the environmental stimuli of the cells. Addiction is a multifactorial and polygenic condition; therefore, various factors and interactions must be considered in the study of epigenetics in addiction. For future studies, it is recommended to use a higher dose of drugs with different time courses to clarify the effect of addictive drugs on epigenetic changes.
Ethical Considerations
Compliance with ethical guidelines
All study procedures complied with the ethical guidelines of the Declaration of Helsinki 2013.
Funding
This research was part of the MSc. thesis of the first author, Maedeh Mohammad Alizadeh, (No: 9410279) in Cellular and Molecular Research Center, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran.
Authors contributions
Design of the study: Parvaneh Keshavarz, Parvin Babaei; Collection of samples, and experimental procedure: Maedeh Mohammad Alizadeh; Interpretation of data, assembly, critical revision of the article for important intellectual content, and final approval of the study: Seyed Reza Kazemi Nezhad, Parvin Babaei, Parvaneh Keshavarz; Writing the article and its editing: Maedeh Mohammad Alizadeh, and Parvaneh Keshavarz.
Conflict of interest
The authors declared no conflict of interest.
Acknowledgements
We appreciate the assistance of Guilan University of Medical Sciences and Shahid Chamran University of Ahvaz, Iran.
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Type of Study: Research | Subject: Special
Received: 2019/05/16 | Accepted: 2019/08/27 | Published: 2019/10/1

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