{"paper_id":"01034a01-c547-45f2-a681-24e3c1096a10","body_text":"Gene Expression Study of Some Addictive Genes in the Hippocampal Region of Albino Rats Treated with a Brand of Energy Drink at Sub-Chronic Period | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Gene Expression Study of Some Addictive Genes in the Hippocampal Region of Albino Rats Treated with a Brand of Energy Drink at Sub-Chronic Period Yahaya Hassan Ahmed Gandawa, Fatima SHETTIMA Dibal, Garba Uthman Sadiq, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7134304/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Addiction to energy drinks (ED) is a growing concern, with potential impacts on neurobiology and gene expression in the hippocampal region. This study examines the impact of Fearless energy drink (FED) on the expression of addiction-related genes within the hippocampal region of albino rats following sub-chronic exposure. Twenty-four (24) adult albino rats were acclimatised and were randomly assigned into four groups: A, B, C, and D (n = 6). Group A served as the control group, whereas Groups B, C, and D received oral administration of Fearless energy drink via oropharyngeal gavage at daily doses of 7 ml/kg, 14 ml/kg, and 21 ml/kg, respectively, for a continuous period of sixty days. At the end of the administration, brain tissue was excised for molecular studies, and gene expression was analysed using quantitative real-time polymerase chain reaction (qRT-PCR). The data were analysed following the manufacturer's instructions for the Real-time PCR instrument. Results The gene expression analysis focused on three genes of interest: The dopamine D 2 receptor (DRD 2 ), catechol-O-methyl transferase (COMT), and Mu-1 opioid receptor (OPRM1), all of which are associated with addictive behaviour. These target genes exhibited differential expression levels across the various treatment groups. Notably, the treated samples exhibit varying degrees of down-regulation compared to the control, as indicated by negative delta Ct (∆ Ct ) values. This study suggests that the treatment had an impact on suppressing the expression levels of the addictive genes, highlighting the potential impact of the fearless Energy drink on the regulation of the addictive genes of interest. Conclusion The study demonstrates that Fearless energy drink causes a dose-dependent suppression of addiction-related genes (DRD 2 , COMT, and OPRM1) in the hippocampus of albino rats. These findings suggest that chronic consumption of energy drinks may disrupt normal neurobiological gene regulation and could contribute to addiction-related changes in the brain. Energy drink Gene expression Albino rats down-regulation Addictive genes Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Energy drinks (ED) are non-alcoholic, often minimally carbonated fortified beverages, similar to soft drinks, and are commonly consumed to enhance energy, alertness, and concentration (Muxiddinovna, 2022; Cadoni and Peana, 2023). Energy drinks first appeared in Europe and Asia in the 1960s in response to consumer demand for a dietary supplement that would result in increased energy (Erdmann et al ., 2021). They contain ingredients intended to enhance mental and physical performance, such as caffeine as the primary component (Tabassum et al ., 2021), along with natural substances like taurine, guarana, and ginseng (Olatona et al ., 2018), sugar, vitamins B, and herbal extracts. Most of these substances are not included on the list approved by the U.S. Food and Drug Administration (FDA). However, the levels of these substances vary among different brands of EDs, and in most cases, are higher than allowable values (Mansy et al ., 2017). In Nigeria, the National Agency for Food and Drug Administration and Control (NAFDAC) has warned citizens against the consumption of highly caffeinated energy drinks, noting that many of these products are sold and distributed without a NAFDAC registration number (Jesupemi, 2023). The use and abuse of EDs are increasing continuously worldwide, and they are consumed frequently by young people aged between 13 and 35 for their psychoactive, stimulant, and performance-enhancing properties (Vargiu et al., 2021). More than half of young adults in Lagos State, Nigeria, consume at least one can of energy drink per month, while approximately 6% consume energy drinks daily (Olatona et al ., 2018). The majority of the users are male, and they are unaware of the amount of caffeine contained in energy drinks (Bano et al ., 2020). Addictive genes are genes that are involved in making an individual more or less vulnerable to addiction to certain substances such as caffeinated energy drinks, tramadol, alcohol and other related substances. These genes are also involved in altering various aspects of brain chemistry, reward processing, and impulse control, which in turn can affect susceptibility to addiction (Jordan and Xi, 2022). The following are some examples of addictive genes; Addictive genes ( DRD 2 , COMT and OPRM1 ) are more common in people addicted to alcohol, cocaine, cannabis, caffeine, and opioids. The variation likely affects how drugs influence the reward pathway (mesolimbic dopamine pathway). Addictive genes refer to biological differences that may make a person more or less susceptible to addiction (Genetic Science Learning Centre, 2020). Scientists are now studying how genes can play a role in making a person vulnerable to drug addiction, or in protecting a person against drug addiction and continued study of genetic factors in drug addiction can provide new ways for understanding the disease of drug addiction, and can lead to new therapies for preventing and treating it (National Institute on Drug Abuse, 2020). There are multiple genes associated with addiction, as well as genes associated with addiction to specific substances or drugs (American Addiction Centre, 2023). There are few or limited studies to address the potentially harmful effects of these EDs on the expression of some addictive genes in the hippocampal region of albino rats, and the risk of intense intake of these EDs amidst athletes, students and young adults has largely gone out of control and is becoming a global challenge by affecting public health and economics. MATERIAL AND METHOD Reagents Formaldehyde (10% formalin), Cryovials, Bouins’ fluid, RNA extraction agents (Trizol) reagent, Phosphate-buffered saline (PBS), Chloroform, Isopropanol, 70% ethanol, Nuclease free water (NFW), RNAlater, Dimethyl Sulfoxide (DMSO), Protease Inhibitors, Lysis Buffers, Reverse Transcription Reagents, qPCR Master mix, RNA/DNA extraction and purification kit, Luna one-step RT-qPCR kit, RNA Extraction Reagents, cDNA Synthesis Kit, Primers and Probes, Specific Gene primers (DRD2, COMT, and OPRM1). Instruments Beakers, syringes (1ml, and 2ml), NG-Tube, weighing balance, hand Gloves, cotton wool, stop watch, permanent markers, meter rule, threads, Real-time qPCR machine, Nano drop machine, -50 0 C refrigerator, centrifuge machine, homogeniser machine, and micro pipette, Thermal cycler (PCR Machine), Reaction Tubes or Plates, Microcentrifuge Tubes, Pipettes and Tips, Electrophoresis System, Scissors, Scalpel, Forceps, Dry Ice and Petri Dish on Ice. All the total RNA purification kits were purchased from Norgen Biotek Corporation based in Canada, with company address 3430schmon parkway. Thorold, Ontario, Canada L2V 4Y6. Experimental Site The Animal Research and Ethical Committee (AREC) on Animal Use of the Faculty of Pharmacy, University of Maiduguri, approved the commencement of this research. The research was carried out at two different sites. The first phase of the experiment was conducted in the Pharmacology and Toxicology Laboratory, Faculty of Pharmacy, University of Maiduguri, where the substance was administered to albino rats for a period of sixty (60) days as a sub-chronic study (Charles River, n.d.). The second phase was carried out at the Northeast Zonal Biotechnology Centre of Excellence, University of Maiduguri, for the molecular analysis of gene expression. Animal treatment Twenty-four adult albino rats, 3-4 months old of both sexes with an average weight of (90-120 g) were obtained and purchased from Faculty of Pharmacy Animal House Bayero University Kano, Kano State Nigeria and brought to Department of Pharmacology and Toxicology, Faculty of Pharmacy animal house where the animals were maintained for acclimatisation for two weeks before the experiment. The rats were randomly assigned to four groups: A, B, C, and D (n = 6), and were kept in separate cages. Group A served as the control, while Groups B, C, and D received low, medium, and high doses of the Fearless energy drink, respectively. The animals had free access to distilled water and were fed grower’s mash (TOPFEEDS), produced by Premier Feed Mills Co. Ltd, and purchased from Maiduguri Monday Market, Borno State, for the duration of the study. Before the commencement of the experiment, all rats were weighed and the values recorded; each rat in every group was numbered and marked on the tail with a permanent marker for easy identification. Calculation and Administration of the Fearless Energy Drink FED The Fearless energy drinks (FED), a product of Rite Foods Ltd, located along the Shagamu–Benin Expressway, Ososa, Ogun State, Nigeria, with a production date of 02/12/2022 and an expiry date of 02/06/2023, were purchased from retail outlets in the commercial area of the University of Maiduguri. The volume of Fearless energy drink to be administered to each rat was calculated based on the average intake of a 70 kg adult male (one 500 ml bottle of Fearless) (Ferreira et al ., 2004). A bottle of Fearless ED was considered as (low dose), two bottles as the (medium dose), and three bottles as the (high dose) (Sobel 2025). The rats in the control group were given distilled water only liberally for a duration of 60 days, in line with an improved method (Adjene et al ., 2010). The dose of the energy drink was chosen based on the protocol described by Akande and Banjoko (2011), the dose was modified and then was given through oral gavage with the aid of oropharyngeal tubes at dose of (7 ml/kg), (14 ml/kg), and (21 ml/kg), to the rats in Groups B, C and D, respectively, daily for sixty days. Tissue Excision At the end of the administration, all the rats were anesthetised via intraperitoneal injection of 0.5 ml ketamine hydrochloride (Ketaset) manufactured Zoetis, the rats were sacrificed through cervical dislocation on the 61 st day of the experiment, full organ dissection was carried out using (scissors, scalpel and forceps) and after organ dissection, the dissected brains were all harvested, rinsed in Phosphate Buffer Saline (PBS), placed in the petri dish on ice, the hippocampus was carefully isolated from the surrounding brain regions, the dissected hippocampus was transferred into microcentrifuge tube containing cold PBS, it was rinsed gently to remove any blood or debris. The dissected hippocampus samples were snap-frozen, stored in a refrigerator at -80 °C for further analysis of molecular studies that focused on gene of interest [Dopamine receptor gene ( DRD 2 ), Catechol-O-methyl transferase gene ( COMT ), and Opioid receptor Mu-1 gene ( OPRM1 )]. Molecular Studies Optimisation of the Real-Time qPCR Before the evaluation of the target samples (both control and treated groups), optimisation of the polymerase chain reaction (PCR) conditions, as described by Zhao et al . (2021), was carried out to enhance performance and minimise the risk of failure. This step was also essential for determining the optimal annealing temperature and validating the primer design. Extraction of Total Ribonucleic acid (RNA) Cell lysate preparation: The tissue sample from the animal was excised and homogenised by the addition of 600 μL of Buffer RL, followed by cell lysis to release the RNA. Binding of RNA to column : The RNA was prepared for column binding by adding 600 μL of the lysate, followed by centrifugation for 1 minute at 14,000 × g (approximately 14,000 RPM) Column wash : The column was washed by applying 400 μL of Wash Solution A to remove salts and other contaminants before elution, followed by centrifugation for 1 minute. This process was repeated two to three times. 3.4.2.4 Elution of the RNA : The RNA was eluted by adding 50 μL of Elution Solution A to the column, followed by centrifugation for 2 minutes at 2,000 × g (approximately 2,000 RPM), and then for 1 minute at 14,000 × g (approximately 14,000 RPM). The volume of the eluted RNA was recorded. 3.4.2.5 Storage of RNA : The purified RNA samples were stored at -20°C for 7 days. Data Analysis The data obtained from this study were analysed in accordance with the real-time qPCR instrument manufacturer's instructions, as outlined below: Calculation of the Ct values : The Ct values of both the target gene(s) and reference gene(s) obtained from the qPCR machine were recorded. Calculation of ΔCt ( Delta Ct) : This was achieved by subtracting the Ct value of the reference gene(s) from the Ct value of the target gene(s) for each sample. Calculation of ΔΔCt ( delta delta Ct) : This was determined by subtracting the average delta the Ct value of the control sample from the delta Ct values of each experimental sample. Calculation of Fold Change : Finally, the 2^–(ΔΔCt) formula was used to calculate fold change, indicating the relative expression level of the target genes compared to the reference genes in the experimental samples. RESULT Expression of the dopamine D2 receptor gene ( DRD 2 ): In the control group, the ∆Ct value was –13.48, serving as the reference point for comparison. In the treatment groups, the ∆Ct value was –6.17, indicating a less pronounced down-regulation (i.e., a reduction in the production and quantification of cellular components such as RNA and protein by the cell in response to a stimulus) at the low dose when compared to the control group. The medium-dose group exhibited a ∆ Ct value of -7.42, reflecting a moderate down-regulation compared to the control group. Notably, the high-dose treatment group exhibited the most substantial change in gene expression, with a ∆Ct value of –12.77, suggesting significant down-regulation. The low-dose treatment resulted in a minimal 2^–(∆∆Ct) value of 0.01, indicating a marginal decrease in DRD2 expression compared to the control group, which had a 2^–(∆∆Ct) value of 1.00. The medium-dose treatment exhibited a slightly higher 2^- (∆∆ Ct) value of 0.02, signifying a modest reduction in DRD 2 expression compared to the control. Remarkably, the high-dose treatment showed a 2^- (∆∆ Ct) value of 0.61, indicating a substantial decrease in DRD 2 expression compared to control (Table 1 and Figure 1). Table 1: Expression of dopamine D 2 receptor gene ( DRD 2 ) Sample A-Ct ∆ Ct ∆∆ Ct 2^-(∆∆ ct) GOI(DRD2) HK Control 26.46 39.93 -13.48 0.00 1.00 Treated at low dose 23.47 29.64 -6.17 7.31 0.01 Treated at medium dose 24.72 32.14 -7.42 6.06 0.02 Treated at high dose 21.96 34.73 -12.77 0.71 0.61 Keys: A-Ct= Average Ct value, GOI= Gene of interest, HK= House Keeping Gene, ∆ Ct = delta Ct value, ∆∆ Ct = delta delta Ct value and 2^-(∆∆ ct) = Fold Change. Expression of the catechol-O-methyl transferase gene ( COMT ): In the control group, the expression level was 26.410 with a ∆Ct of –13.520. In the treated groups, the expression levels at low, medium, and high doses were 23.765, 25.530, and 28.395, respectively. The housekeeping gene (HK) showed a stable expression level across all samples. The comparison of gene expression levels (∆ Ct) revealed a down-regulation in the treated groups compared to the control. Particularly, at low, medium, and high doses, the ∆ Ct values were -5.870, -6.610, and -6.330, respectively (Table 2). The 2^-(∆∆ Ct) values of the control are 1.000, serving as reference points for comparison. The corresponding 2^- (∆∆ Ct) values at treated groups demonstrated a dose-dependent response. As the dose increased, there was a consistent reduction in COMT expression, with 2^–(∆∆Ct) values of 0.005, 0.008, and 0.007, respectively, compared to the control (Table 2 and Figure 2). Table 2: Expression of catechol-O-methyl transferase gene (COMT) Sample A-Ct ∆ Ct ∆∆ Ct 2^-(∆∆ ct) GOI(COMT) HK Control 26.410 39.930 -13.520 0.000 1.000 Treated at low dose 23.765 29.635 -5.870 7.650 0.005 Treated at medium dose 25.530 32.140 -6.610 6.910 0.008 Treated at high dose 28.395 34.725 -6.330 7.190 0.007 Keys: A-Ct= Average Ct value, GOI= Gene of interest, HK= House Keeping Gene, ∆ Ct = delta Ct value, ∆∆ Ct = delta delta Ct value and 2^-(∆∆ ct) = Fold Change. Expression of mu-1 opioid receptor (OPRM1) gene : the control group serves as the baseline for comparisons, with a Gene of interest (GOI) expression of (20.8750) and housekeeping gene expression of 39.9300. The ∆ Ct is (-19.0550), indicating the baseline difference between the GOI and HK. The low-dose treated group showed a decrease in the expression of the gene of interest (GOI), with a significant ∆∆Ct value of 12.6300 and a corresponding 2^–(∆∆Ct) value of 0.0002, indicating notable down-regulation . Similarly, the medium- and high-dose groups both exhibited consistent and significant down-regulation , with identical ∆∆Ct values of 9.0850 and 2^–(∆∆Ct) values of 0.0018, compared to the control (Table 3 and Figure 3). Table 3: Expression of mu-1 opioid receptor (OPRM1) gene Sample A-Ct ∆ Ct ∆∆ Ct 2^-(∆∆ ct) GOI(OPRM1) HK Control 20.8750 39.9300 -19.0550 0.0000 1.0000 Treated at low dose 23.2100 29.6350 -6.4250 12.6300 0.0002 Treated at medium dose 22.1700 32.1400 -9.9700 9.0850 0.0018 Treated at high dose 24.7550 34.7250 -9.9700 9.0850 0.0018 Keys: A-Ct= Average Ct value, GOI= Gene of interest, HK= House Keeping Gene, ∆ Ct = delta Ct value, ∆∆ Ct = delta delta Ct value and 2^-(∆∆ ct) = Fold Change. DISCUSSION The results of the present study confirm that sub-chronic (60-day) administration of varying doses of Fearless Energy Drink (FED) altered the expression levels of the dopamine D 2 receptor ( DRD 2 ) in the hippocampal region of the brains of albino rats. The gene of interest (GOI), DRD 2 , demonstrated different expression levels across different treatment groups. Remarkably, the treated groups display varying degrees of down-regulation compared to the control, as indicated by negative ∆ Ct values. Pharmacologically, the down-regulation of the DRD 2 gene, which encodes dopamine D 2 receptors, may result in reduced receptor availability, potentially leading to an increase in dopamine release. This could result in elevated levels of dopamine in the synaptic cleft (Noble, 2000 ). This is in agreement with growing evidence from several studies that suggests caffeine intoxication from EDs, and that EDs may serve as a gateway to other forms of drug dependence (Griffiths et al ., 2019). Another study reported that coffee extract and its components, such as caffeine, may influence gene expression by altering histone modifications, which are essential for regulating the physical properties of chromatin and its corresponding transcriptional state, DNA methylation, and non-coding RNA (ncRNA) expression (Ding et al., 2023 ). Similarly, a separate study found that a psychostimulant such as caffeine induced down-regulation of DRD 2 mRNA expression in the testes of rats (Gonzalez et al., 2015 ). The gene expression study for catechol-O-methyl transferase ( COMT ) analysed the expression of the GOI, COMT , across different samples, including a control group and treated samples at low, medium, and high doses. The FED consumption revealed that the COMT gene exhibited differential expression levels across the experimental groups. This implies that the treatment influenced down-regulation the expression of the addictive gene COMT . As the dose increases, there is a consistent decrease in COMT expression. The COMT gene encodes catechol-O-methyltransferase, an enzyme involved in the breakdown of dopamine. The down-regulation of the COMT gene could lead to reduced activity of this enzyme, potentially resulting in decreased dopamine metabolism. As a result, there might be an accumulation of dopamine in the synaptic cleft, prolonging its presence and enhancing neurotransmission (Simpson et al., 2014 ; Kennedy et al., 2015 ). Caffeinated energy drinks may indirectly affect the COMT enzyme, potentially influencing dopamine levels. However, the relationship is complex, and the precise mechanisms remain incompletely understood. It was also noted that the gene expression study for mu-1 opioid receptor ( OPRM1 ) gene reveals alteration in the expression of the GOI: OPRM1 under different treatment samples compared to the control. The data include the expression of the gene of interest ( OPRM1 ), the housekeeping gene (HK), and the corresponding ∆Ct values. The 2^- (∆∆ Ct) (fold change value) proposes a significant down-regulation in gene expression, which emphasises the potential influence of the fearless energy drink on the regulation of the OPRM1 gene. The down-regulation induced by FED in the present findings on the OPRM1 gene could potentially impact the opioid system, leading to alterations in pain perception, mood regulation, reward processing, and possibly vulnerability to conditions involving dysregulation of the opioid and dopamine systems, such as addiction (Picci et al., 2022 ). The result of the current study agreed with some findings of the gene expression studies that caffeinated energy drinks induced alteration of gene expression in various organ of the body like that of El-Terras et al. ( 2016 ), reported that carbonated soft drinks contained caffeine, were also observed to down-regulation the expression of monoaminoxidase-A ( MAO-A ) and acetylcholinesterase (AchE) serum and mRNA level in the brain. Additionally, there was an up-regulation of mRNA expression of the dopamine D 2 receptor ( DRD 2 ), while the expression of the 5-hydroxytryptamine transporter ( 5HTT ) was decreased (El-Terras et al., 2016 ). Additional findings of the gene expression study reported that genes involved in neurogenesis were decreased, which could be related to the impairment of memory function. Ethanol significantly altered the expression of 416 out of 11,727 genes expressed in the ventral hippocampus. While in the medial prefrontal cortex, 638 out of 11,579 were altered; genes in cellular stress and inflammatory pathways were increased, as were genes involved in oxidative phosphorylation (McClintick et al., 2018 ). Caffeine is a central nervous system stimulant and can interact with various genes and neurotransmitter systems. However, its primary mode of action involves adenosine receptors rather than direct modulation of DRD 2 , COMT , or OPRM1 genes. The dopamine neurotransmitter plays a crucial role in addiction by reinforcing behaviours associated with pleasure or reward. High levels of dopamine can contribute to the development of addiction by reinforcing the desire to repeat actions that lead to pleasure, such as drug use, alcohol intake or certain behaviours like gambling and heavy activities. A 500 ml bottle of Fearless Energy Drink (FED) contains approximately 155 mg of caffeine, which is proposed to be responsible for the observed changes in gene expression levels, due to the high caffeine content of energy drinks (Al-Shaar et al., 2017 ). However, the concentration of the caffeine content in the fearless energy drink singlehandedly cannot induced the observed changes (Al-Basher et al., 2018 ), but a combination of the caffeine and other active stimulants present in the energy drink like taurine, guarana and herbal extracts were proposed to potentiates the observed changes (Zeidan-Chulia et al., 2013 ). CONCLUSION The gene expression findings concluded that the three addictive genes of interest were dose-dependently down-regulated in response to FED, with higher doses exhibiting the most pronounced effects, resulting in a substantial reduction in the expression levels of the genes studied. However, the down-regulatory effect of FED was more pronounced on the DRD2 gene than on the COMT gene, and similarly, more pronounced on the COMT gene than on the OPRM1 gene. Recommendation Based on the observed dose-dependent down-regulation of the three addictive genes of interest following administration of Fearless Energy Drink, further investigations are recommended to: Explore the specific mechanisms underlying the observed alteration of the gene expression. Validate the results using additional experimental approaches or biological assays. Validate these findings using complementary techniques and possibly expand the study to include other relevant genes or factors that could contribute to the observed effects. Collaborate with experts in the field to gain insights into the broader context of the observed gene expression changes. Abbreviations FED: Fearless energy drink; ED: Energy drink; DRD 2 : Dopamine receptor D 2 subtype; SUD: Substance use disorder; PTSD: Post-traumatic stress disorder; COMT : catechol-O-methyltransferase; OPRM1 : Opioid receptor Miu-1; PBS: Phosphate-buffered saline; NFW: Nuclease free water; DMSO: Dimethyl Sulfoxide; NG-Tube: Nasogastric tube; AREC: Animal Research and Ethical Committee; DNA: Deoxyribonucleic acid; RNA: Ribonucleic acid; RPM: Rotation per minutes; PCR: Polymerase chain reaction; HK: Housekeeping gene; GOI: Gene of interest; A-Ct: Average Ct value; Ct: delta Ct value; ∆∆ Ct: delta delta Ct value; 2^-(∆∆ ct): Fold Change; MAO-A : Monoaminoxidase-A; AchE: Acetylcholinesterase; CA1: Cornu ammonis-1; DG: Dentate gyrus. Declarations Acknowledgement We thankfully acknowledged the contribution of Prof. Usman Sadiq, the Dean of the Faculty of Pharmacy, Unimaid, and Prof. Bala Usman Shamaki of Veterinary Pharmacology, Faculty of Veterinary Medicine. Unimaid, for their support, supervision, and guidance during this research. Author contribution YHAG, FSD, GUS, and TSY initiated the work, YHAG and FSD carried out the laboratory animal handling and treatment, while GUS supervised the work and TSY analysed the data. YHAG and FSD wrote the draft of the manuscript. GUS revised the draft, while all authors approved the final version of the work before submission to the journal. Funding No financial support was received for this research. Availability of data and materials The corresponding author will be provided with data upon request. Ethics approval and consent to participate All experimental procedures were carried out in strict accordance with the Guide to the Care and Use of Laboratory Animals in Research and Teaching, as detailed in NIH publications (Council 2011). Consent for publication Not applicable. Competing interests The authors declare no conflict of interest. Author details Department of Pharmacology and Toxicology. Faculty of Pharmacy, University of Maiduguri, Maiduguri, Borno State, Nigeria. References Adjene, J.O., Ezeoke, J.C., Nwose, E.U. (2010). Histological effects of chronic consumption of soda pop drinks on kidney of adult Wistar rats. North America Journal of Medical Science, 2(1): 215‑217. Akande, I.S. and Banjoko, O.A. (2011). Assessment of biochemical effect of “power horse” energy drink on hepatic, renal and histological functions in sprague dawley rats. Annual review & research in biology , 1(3), 45-56. Al-Basher, G.I., Aljabal, H., Almeer, R., Allam, A.A. and Mahmoud, A.M. (2018). Perinatal exposure to energy drink induces oxidative damage in the liver, kidney and brain, and behavioral alterations in mice offspring. Biomedical Pharmacotherapy , 102: 798-811. Al-Shaar, L., Vercammen, K., Lu, C., Richard- Son, S., Tamez, M. And Mattei, J. (2017). Health effects and public health concerns of energy drink consumption in the United States: A Mini-Review. Front. Public health , 5: 225. American Addiction Centre, (2023). Is drug addiction genetic? Retrieved 4 th January, 2024 from . Bano, S.S., Shabana, A., Saddaf, A., Ali, A., and Imran, B. (2020). Effects of caffeinated energy drink withdrawal on effects of caffeinated energy drink withdrawal on histological and biochemical parameters of adult histological and biochemical parameters of adult albino rat kidneys. Journal of Medicinal Science , 28(2), 107-111. Cadoni, C. and Peana, A.T. (2023). Energy drinks at adolescences: awareness or unawareness. Frontiers in Behavioural Neuroscience , 2023: 1-8. Charles river, (n.d) Sub-chronic and chronic toxicity studies. Retrieved on 4 th February, 2024, from Ding, Q., Xu, Y., and Lau, A.T.Y. (2023). The epigenetic effects of coffee. Journal Molecules , 28(4), 1770. El-Terras, A., Mohammed, M.S., Alkhedaide, A., Fouad, A.H., Alharthy, A., and Elah, A.B. (2016). Carbonated soft drinks induced oxidative stress and altered expression of certain genes in the brain of wistar rats. Molecular Medicine Reports , 13(4), 3147-3154. Erdmann, J., Wiciński, M., Wódkiewicz, E., Nowaczewska, M., Słupski, M., Otto, S.W., Kubiak, K., Huk-Wieliczuk, E. and Malinowski, B. (2021). Effects of Energy Drink Consumption on Physical Performance and Potential Danger of Inordinate Usage. Nutrients , 13: 2506. . Ferreira, E.S., Hartmann Quadros, M.I., Trindade, A.A., Takahashi, S., Koyama, G.R., and Souza-Formigoni, O.M. (2004). Can energy drinks reduce the depressor effect of ethanol? An experimental study in mice. Physiology and Behaviour , 82(5), 841-847. Genetic Science Leaning Centre, (2020). Genes and addiction. Retrieved January 02, 2024, from . Gonzalez, C.R., Gonzalez, B., Matzkin, M.E., Muniz, J.A., Cadet, J.L., Garcia-Rill, E. (2015). Psychostimulant induced testicular toxicity in mice; evidence of cocaine and caffeine on the local dopaminergic system. Plos One , 10(11), 0142713. Griffiths, R.R., Chad, J.R., Erick, C.S. (2009). Caffeinated energy drink. A growing problem. Journal of Drug and Alcohol Dependence , 99(3), 1-10. Jesupemi, A. (2023). Alert: NAFDAC warns against consumption of energy drink brand. TheCable , (November 3, 2023), retrieved from https//www.thecable.ng on January 5, 2024. Jordan, C.J and Xi, X (2022). Identification of the risk genes associated with vulnerability to addiction: major findings from transgenic animals. Front. Neurosci., 15 (2021). Kennedy, J.L., Arqam, Q., Clement, C.Z., Yuko, H., Arun, K.T., Sheraz, C., Behdin, N., and Joseph, H.B. (2015). The role of the catechol-o-methyltransferase (COMT) gene val158met in aggressive behaviour, a review of genetic studies. Current Neuropharmacology , 13, 802-814. Mansy, W., Deema, M.A., Mona, H., Enas, Z. (2017). Effects of chronic consumption of energy drinks on liver and kidney of experimental rats. Tropical Journal of Pharmaceutical Research , 16 (12): 2849-2856. McClintick, J.N., McBride, W.J., Bell, R.L., Dina, Z., and Liu, Y. (2018). Gene expression changes in the ventral hippocampus and medial prefrontal cortex of adolescent alcohol-preferring (p) rats following binge-like alcohol drinking. Journal of Alcohol , 68, 37-47. Muxiddinovna, I.M. (2022). Effects of energy drinks on biochemical and sperm parameters in albino rats. Central Asian Journal of Medical and Natural Sciences , 3(3): 2660-4159. National Institute on Drug Abuse, NIDA. (2020). Drugs, brain, and behaviour: the science of addiction drug misuse and addiction. Retrieved September 5 th 2023 from . Noble, P.E (2000). The DRD2 in psychiatric and neurological disorders and its phenotypes. Pharmacogenomics , 1(3), 309-333. Olatona, F.A., Ijeoma, O.A., Sunday, A.A., and Temitope, W.L.A. (2018). Energy drinks consumption among football players in Lagos, Nigeria. South African Journal of Clinical Nutrition , 31(4), 84–88. Picci, G., Fishbein, D.H., Vanmeter, J.W., Rose, E.J. (2022). Effects of OPRM1 and DRD2 on brain structure in drug-naïve adolescents: genetic and neural vulnerabilities to substance use. Psychopharmacology (Berl) , 239(1): 141-152. Simpson, E.H., Morud, J., Winiger, V., Biezonski, D., Zhu, J.P., Bach, M.E., Malleret, G., Polan, H.J., Ng-Evan, S., Phillip, P.E.M., Kellendonk, C. and Kandel, E.R. (2014). Genetic variation in COMT activity impacts learning and dopamine release capacity in the striatum. Learning and Memory , 21(4), 205-214. Sobel, A. (2025). How many energy drinks a day is safe?. . Published online on 24 th June, 2025. Retrieved online on 5 th July, 2025. Tabassum, Y., Maheen, H., Khan, B., Zafar, I.B., Qamar, J., Abdul, R. and Muhammad, U. (2021). Constructive effects of energy drink consumption on players’ performance. Palarch`s Journal of Archaeology Egypt/Egyptology , 18(8), 4099-4107. Vargiu, R., Francesca, B., Carla, L., Daniele, L., Alessandro, C., Pier, P.B. and Valentina, B. (2021). Chronic red bull consumption during adolescence: Effect on mesocortical and mesolimbic dopamine transmission and cardiovascular system in adult rats. , 2021, 14, 609. Zeidan-Chulia, F., Gelain, D.P., Kolling, E.A., Rybarczyk-Filho, J.L., Ambrosi, P., Resende, T.S. (2013). Major components of energy drinks (caffeine, taurine, and guarana) exert cytotoxic effects on human neuronal SH-SY5Y cells by decreasing reactive oxygen species production. Oxid. Med. Cell . ID791795. Zhou, F., Maren, A.N., Pawel, Z., Liao, Y.K., Lu, H., Duduit, R.J., Huang, D., Ashrafi, H., Zhao, T., Hueeta, I.A., Ranney, G.T., and Liu, W. (2021). An optimize protocol for stepwise optimization of real-Time RT-PCR analysis. Horticulture Research , 8, 179. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-7134304\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":496633447,\"identity\":\"b41ab652-70ba-453b-9483-6625abb7aec5\",\"order_by\":0,\"name\":\"Yahaya Hassan Ahmed Gandawa\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYDACCQglx8DA2MDAYMAMZCcQp8WYdC2JDRCaCC3m0s3PPt2oqU3fcLu5dcOPAmsGfvYcA+aCX7i1WM45Zjw759jx3A13Drbd7DFIZ5DseWPAPLMPtxaDGwnGzDlsx3I33Ehsu8FjcBgoArSFtweflvTPzDn/jqUbALXc/APUYk9YS44xc25bTQJIy22wLRJALTw/8PhlRk4xc27fAcOZIC0yBuk8EmeeFRzmbcCtxVwifTNzzrc6eb4b6c9uvvljLcffnrzxMc8fPA6DUIfhAjwg4gBjG0EtdejieGwZBaNgFIyCEQcAaNNWyXzBEMcAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"University of Maiduguri\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Yahaya\",\"middleName\":\"Hassan Ahmed\",\"lastName\":\"Gandawa\",\"suffix\":\"\"},{\"id\":496633448,\"identity\":\"2b9cbc7f-49c1-4bb9-9773-d8afe9446a87\",\"order_by\":1,\"name\":\"Fatima SHETTIMA Dibal\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Maiduguri\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Fatima\",\"middleName\":\"SHETTIMA\",\"lastName\":\"Dibal\",\"suffix\":\"\"},{\"id\":496633449,\"identity\":\"6af9d43c-6f44-450b-9f70-187ac7066f23\",\"order_by\":2,\"name\":\"Garba Uthman Sadiq\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Maiduguri\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Garba\",\"middleName\":\"Uthman\",\"lastName\":\"Sadiq\",\"suffix\":\"\"},{\"id\":496633450,\"identity\":\"c5568e03-0d70-42ac-963b-a2352bbe18e9\",\"order_by\":3,\"name\":\"Timothy Samuel Yerima\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Maiduguri\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Timothy\",\"middleName\":\"Samuel\",\"lastName\":\"Yerima\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-07-15 23:08:12\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-7134304/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-7134304/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":88560713,\"identity\":\"3d62edf3-868e-4a1c-bdae-ff0d1d6e75b5\",\"added_by\":\"auto\",\"created_at\":\"2025-08-07 17:55:46\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":33961,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFold change of dopamine D\\u003csub\\u003e2\\u003c/sub\\u003e receptor (\\u003cem\\u003eDRD\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e) gene against the control group\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7134304/v1/3af5ac98383609437ae8d221.jpg\"},{\"id\":88560079,\"identity\":\"7d56e571-5779-4814-9b31-3173d3dcaedf\",\"added_by\":\"auto\",\"created_at\":\"2025-08-07 17:47:46\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":33133,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFold change of COMT gene against the control group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7134304/v1/0d0a0fec33824e70acfd3e00.jpg\"},{\"id\":88560715,\"identity\":\"670686f4-28cf-4faf-9f08-386c5cb76411\",\"added_by\":\"auto\",\"created_at\":\"2025-08-07 17:55:46\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":32112,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFold change of mu-1 opioid receptor (OPRM1) gene against the control group\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7134304/v1/94afbfc3100a2d5654ae2eb1.jpg\"},{\"id\":92390306,\"identity\":\"d6f3894e-831d-4e70-aa40-620c81747d28\",\"added_by\":\"auto\",\"created_at\":\"2025-09-29 08:24:24\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1217708,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7134304/v1/4409e08b-35b6-47e9-b63f-6e46b3a39b03.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"\\u003cp\\u003eGene Expression Study of Some Addictive Genes in the Hippocampal Region of Albino Rats Treated with a Brand of Energy Drink at Sub-Chronic Period\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"INTRODUCTION\",\"content\":\"\\u003cp\\u003eEnergy drinks (ED) are non-alcoholic, often minimally carbonated fortified beverages, similar to soft drinks, and are commonly consumed to enhance energy, alertness, and concentration (Muxiddinovna, 2022; Cadoni and Peana, 2023).\\u0026nbsp;Energy drinks first appeared in Europe and Asia in the 1960s in response to consumer demand for a dietary supplement that would result in increased energy (Erdmann \\u003cem\\u003eet al\\u003c/em\\u003e., 2021). They contain ingredients intended to enhance mental and physical performance, such as caffeine as the primary component (Tabassum \\u003cem\\u003eet al\\u003c/em\\u003e., 2021), along with natural substances like taurine, guarana, and ginseng (Olatona \\u003cem\\u003eet al\\u003c/em\\u003e., 2018), sugar, vitamins B, and herbal extracts. Most of these substances are not included on the list approved by the U.S. Food and Drug Administration (FDA). However, the levels of these substances vary among different brands of EDs, and in most cases, are higher than allowable values (Mansy \\u003cem\\u003eet al\\u003c/em\\u003e., 2017). In Nigeria, the National Agency for Food and Drug Administration and Control (NAFDAC) has warned citizens against the consumption of highly caffeinated energy drinks, noting that many of these products are sold and distributed without a NAFDAC registration number (Jesupemi, 2023). The use and abuse of EDs are increasing continuously worldwide, and they are consumed frequently by young people aged between 13 and 35 for their psychoactive, stimulant, and performance-enhancing properties (Vargiu \\u003cem\\u003eet al.,\\u003c/em\\u003e 2021). More than half of young adults in Lagos State, Nigeria, consume at least one can of energy drink per month, while approximately 6% consume energy drinks daily (Olatona \\u003cem\\u003eet al\\u003c/em\\u003e., 2018). The majority of the users are male, and they are unaware of the amount of caffeine contained in energy drinks (Bano \\u003cem\\u003eet al\\u003c/em\\u003e., 2020).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAddictive genes are genes that are involved in making an individual more or less vulnerable to addiction to certain substances such as caffeinated energy drinks, tramadol, alcohol and other related substances. These genes are also involved in altering various aspects of brain chemistry, reward processing, and impulse control, which in turn can affect susceptibility to addiction (Jordan and Xi, 2022). The following are some examples of addictive genes;\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAddictive genes (\\u003cem\\u003eDRD\\u003csub\\u003e2\\u003c/sub\\u003e, COMT\\u003c/em\\u003e and \\u003cem\\u003eOPRM1\\u003c/em\\u003e) are more common in people addicted to alcohol, cocaine, cannabis, caffeine, and opioids. The variation likely affects how drugs influence the reward pathway (mesolimbic dopamine pathway). Addictive genes refer to biological differences that may make a person more or less susceptible to addiction (Genetic Science Learning Centre, 2020). Scientists are now studying how genes can play a role in making a person vulnerable to drug addiction, or in protecting a person against drug addiction and continued study of genetic factors in drug addiction can provide new ways for understanding the disease of drug addiction, and can lead to new therapies for preventing and treating it (National Institute on Drug Abuse, 2020). There are multiple genes associated with addiction, as well as genes associated with addiction to specific substances or drugs (American Addiction Centre, 2023). There are few or limited studies to address the potentially harmful effects of these EDs on the expression of some addictive genes in the hippocampal region of albino rats, and the risk of intense intake of these EDs amidst athletes, students and young adults has largely gone out of control and is becoming a global challenge by affecting public health and economics.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"MATERIAL AND METHOD \",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eReagents\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFormaldehyde (10% formalin), Cryovials, Bouins’ fluid, RNA extraction agents (Trizol) reagent, Phosphate-buffered saline (PBS), Chloroform, Isopropanol, 70% ethanol, Nuclease free water (NFW), RNAlater, Dimethyl Sulfoxide (DMSO), Protease Inhibitors, Lysis Buffers, Reverse Transcription Reagents, qPCR Master mix, \\u0026nbsp;RNA/DNA extraction and purification kit, Luna one-step RT-qPCR kit, RNA Extraction Reagents, cDNA Synthesis Kit, Primers and Probes, Specific Gene primers (DRD2, COMT, and OPRM1).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eInstruments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eBeakers, syringes (1ml, and 2ml), NG-Tube, weighing balance, hand Gloves, cotton wool, stop watch, permanent markers, meter rule, threads, Real-time qPCR machine, Nano drop machine, -50\\u003csup\\u003e0\\u003c/sup\\u003eC refrigerator, centrifuge machine, homogeniser machine, and micro pipette, Thermal cycler (PCR Machine), Reaction Tubes or Plates, Microcentrifuge Tubes, Pipettes and Tips, Electrophoresis System, Scissors, Scalpel, Forceps, Dry Ice and Petri Dish on Ice. All the total RNA purification kits were purchased from Norgen Biotek Corporation based in Canada, with company address 3430schmon parkway. Thorold, Ontario, Canada L2V 4Y6.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eExperimental Site\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe Animal Research and Ethical Committee (AREC) on Animal Use of the Faculty of Pharmacy, University of Maiduguri, approved the commencement of this research. The research was carried out at two different sites. The first phase of the experiment was conducted in the Pharmacology and Toxicology Laboratory, Faculty of Pharmacy, University of Maiduguri, where the substance was administered to albino rats for a period of sixty (60) days as a sub-chronic study (Charles River, n.d.). The second phase was carried out at the Northeast Zonal Biotechnology Centre of Excellence, University of Maiduguri, for the molecular analysis of gene expression.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAnimal treatment\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTwenty-four adult albino rats, 3-4 months old of both sexes with an average weight of (90-120 g) were obtained and purchased from Faculty of Pharmacy Animal House Bayero University Kano, Kano State Nigeria and brought to Department of Pharmacology and Toxicology, Faculty of Pharmacy animal house where the animals were maintained for acclimatisation for two weeks before the experiment. The rats were randomly assigned to four groups: A, B, C, and D (n = 6), and were kept in separate cages. \\u0026nbsp;Group A served as the control, while Groups B, C, and D received low, medium, and high doses of the Fearless energy drink, respectively. The animals had free access to distilled water and were fed grower’s mash (TOPFEEDS), produced by Premier Feed Mills Co. Ltd, and purchased from Maiduguri Monday Market, Borno State, for the duration of the study. Before the commencement of the experiment, all rats were weighed and the values recorded; each rat in every group was numbered and marked on the tail with a permanent marker for easy identification.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCalculation and Administration of the Fearless Energy Drink FED\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe Fearless energy drinks (FED), a product of Rite Foods Ltd, located along the Shagamu–Benin Expressway, Ososa, Ogun State, Nigeria, with a production date of 02/12/2022 and an expiry date of 02/06/2023, were purchased from retail outlets in the commercial area of the University of Maiduguri. The volume of Fearless energy drink to be administered to each rat was calculated based on the average intake of a 70 kg adult male (one 500 ml bottle of Fearless) (Ferreira \\u003cem\\u003eet al\\u003c/em\\u003e., 2004). A bottle of Fearless ED was considered as (low dose), two bottles as the (medium dose), and three bottles as the (high dose) (Sobel 2025). The rats in the control group were given distilled water only liberally for a duration of 60 days, in line with an improved method (Adjene \\u003cem\\u003eet al\\u003c/em\\u003e., 2010). The dose of the energy drink was chosen based on the protocol described by Akande and Banjoko (2011), the dose was modified and then was given through oral gavage with the aid of oropharyngeal tubes at dose of (7 ml/kg), (14 ml/kg), and (21 ml/kg), to the rats in Groups B, C and D, respectively, daily for sixty days.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTissue Excision\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAt the end of the administration, all the rats were anesthetised via intraperitoneal injection of 0.5 ml ketamine hydrochloride (Ketaset) manufactured Zoetis, the rats were sacrificed through cervical dislocation on the 61\\u003csup\\u003est\\u003c/sup\\u003e day of the experiment, full organ dissection was carried out using (scissors, scalpel and forceps) and after organ dissection, the dissected brains were all harvested, rinsed in Phosphate Buffer Saline (PBS), placed in the petri dish on ice, the hippocampus was carefully isolated from the surrounding brain regions, the dissected hippocampus was transferred into microcentrifuge tube containing cold PBS, it was rinsed gently to remove any blood or debris. The dissected hippocampus samples were snap-frozen, stored in a refrigerator at -80 °C for further analysis of molecular studies that focused on gene of interest [Dopamine receptor gene (\\u003cem\\u003eDRD\\u003csub\\u003e2\\u003c/sub\\u003e\\u003c/em\\u003e), Catechol-O-methyl transferase gene (\\u003cem\\u003eCOMT\\u003c/em\\u003e), and Opioid receptor Mu-1 gene (\\u003cem\\u003eOPRM1\\u003c/em\\u003e)].\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMolecular Studies\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eOptimisation of the\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eReal-Time qPCR\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eBefore the evaluation of the target samples (both control and treated groups), optimisation of the polymerase chain reaction (PCR) conditions, as described by Zhao \\u003cem\\u003eet al\\u003c/em\\u003e. (2021), was carried out to enhance performance and minimise the risk of failure. This step was also essential for determining the optimal annealing temperature and validating the primer design.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eExtraction of Total Ribonucleic acid (RNA)\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCell lysate preparation: The tissue sample from the animal was excised and homogenised by the addition of 600 μL of Buffer RL, followed by cell lysis to release the RNA.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eBinding of RNA to column\\u003c/strong\\u003e: The RNA was prepared for column binding by adding 600 μL of the lysate, followed by centrifugation for 1 minute at 14,000 × g (approximately 14,000 RPM)\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eColumn wash\\u003c/strong\\u003e: The column was washed by applying 400 μL of Wash Solution A to remove salts and other contaminants before elution, followed by centrifugation for 1 minute. This process was repeated two to three times.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.4.2.4 Elution of the RNA\\u003c/strong\\u003e: The RNA was eluted by adding 50 μL of Elution Solution A to the column, followed by centrifugation for 2 minutes at 2,000 × g (approximately 2,000 RPM), and then for 1 minute at 14,000 × g (approximately 14,000 RPM). The volume of the eluted RNA was recorded.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.4.2.5 Storage of RNA\\u003c/strong\\u003e: The purified RNA samples were stored at -20°C for 7 days.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData Analysis\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe data obtained from this study were analysed in accordance with the real-time qPCR instrument manufacturer's instructions, as outlined below:\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCalculation of the Ct values\\u003c/strong\\u003e: The Ct values of both the target gene(s) and reference gene(s) obtained from the qPCR machine were recorded.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCalculation of\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eΔCt\\u003c/strong\\u003e (\\u003cstrong\\u003eDelta Ct)\\u003c/strong\\u003e: This was achieved by subtracting the Ct value of the reference gene(s) from the Ct value of the target gene(s) for each sample.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCalculation of\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eΔΔCt\\u003c/strong\\u003e (\\u003cstrong\\u003edelta delta Ct)\\u003c/strong\\u003e: This was determined by subtracting the average delta the Ct value of the control sample from the delta Ct values of each experimental sample.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCalculation of Fold Change\\u003c/strong\\u003e: Finally, the 2^–(ΔΔCt) formula was used to calculate fold change, indicating the relative expression level of the target genes compared to the reference genes in the experimental samples.\\u003c/p\\u003e\"},{\"header\":\"RESULT \",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eExpression of the dopamine D2 receptor gene (\\u003cem\\u003eDRD\\u003csub\\u003e2\\u003c/sub\\u003e\\u003c/em\\u003e):\\u003c/strong\\u003e In the control group, the ∆Ct value was \\u0026ndash;13.48, serving as the reference point for comparison. In the treatment groups, the ∆Ct value was \\u0026ndash;6.17, indicating a less pronounced down-regulation (i.e., a reduction in the production and quantification of cellular components such as RNA and protein by the cell in response to a stimulus) at the low dose when compared to the control group. The medium-dose group exhibited a ∆ Ct\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003evalue of -7.42, reflecting a moderate down-regulation compared to the control group. Notably, the high-dose treatment group exhibited the most substantial change in gene expression, with a ∆Ct value of \\u0026ndash;12.77, suggesting significant down-regulation. The low-dose treatment resulted in a minimal 2^\\u0026ndash;(∆∆Ct) value of 0.01, indicating a marginal decrease in \\u003cem\\u003eDRD2\\u003c/em\\u003e expression compared to the control group, which had a 2^\\u0026ndash;(∆∆Ct) value of 1.00. The medium-dose treatment exhibited a slightly higher 2^- (∆∆ Ct)\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003evalue of 0.02, signifying a modest reduction in \\u003cem\\u003eDRD\\u003csub\\u003e2\\u003c/sub\\u003e\\u003c/em\\u003e expression compared to the control. Remarkably, the high-dose treatment showed a 2^- (∆∆ Ct)\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003evalue of 0.61, indicating a substantial decrease in \\u003cem\\u003eDRD\\u003csub\\u003e2\\u003c/sub\\u003e\\u003c/em\\u003e expression compared to control (Table 1 and Figure 1).\\u003c/p\\u003e\\n\\u003cp\\u003eTable 1: Expression of dopamine D\\u003csub\\u003e2\\u0026nbsp;\\u003c/sub\\u003ereceptor gene (\\u003cem\\u003eDRD\\u003csub\\u003e2\\u003c/sub\\u003e\\u003c/em\\u003e) \\u0026nbsp;\\u0026nbsp;\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"600\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd rowspan=\\\"2\\\" valign=\\\"bottom\\\" style=\\\"width: 189px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eSample\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"bottom\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eA-Ct\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd rowspan=\\\"2\\\" valign=\\\"bottom\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e∆ Ct\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd rowspan=\\\"2\\\" valign=\\\"bottom\\\" style=\\\"width: 85px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e∆∆ Ct\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd rowspan=\\\"2\\\" valign=\\\"bottom\\\" style=\\\"width: 95px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2^-(∆∆ ct)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 99px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eGOI(DRD2)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eHK\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 189px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eControl\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 99px;\\\"\\u003e\\n \\u003cp\\u003e26.46\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e39.93\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e-13.48\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 85px;\\\"\\u003e\\n \\u003cp\\u003e0.00\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 95px;\\\"\\u003e\\n \\u003cp\\u003e1.00\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 189px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTreated at low dose\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 99px;\\\"\\u003e\\n \\u003cp\\u003e23.47\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e29.64\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e-6.17\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 85px;\\\"\\u003e\\n \\u003cp\\u003e7.31\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 95px;\\\"\\u003e\\n \\u003cp\\u003e0.01\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 189px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTreated at medium dose\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 99px;\\\"\\u003e\\n \\u003cp\\u003e24.72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e32.14\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e-7.42\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 85px;\\\"\\u003e\\n \\u003cp\\u003e6.06\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 95px;\\\"\\u003e\\n \\u003cp\\u003e0.02\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 189px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTreated at high dose\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 99px;\\\"\\u003e\\n \\u003cp\\u003e21.96\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e34.73\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e-12.77\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 85px;\\\"\\u003e\\n \\u003cp\\u003e0.71\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 95px;\\\"\\u003e\\n \\u003cp\\u003e0.61\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003eKeys: A-Ct= Average Ct value, GOI= Gene of interest, HK= House Keeping Gene, \\u003cstrong\\u003e∆ Ct\\u003c/strong\\u003e = delta Ct value, \\u003cstrong\\u003e∆∆ Ct\\u003c/strong\\u003e= delta delta Ct value and \\u003cstrong\\u003e2^-(∆∆ ct) =\\u0026nbsp;\\u003c/strong\\u003eFold Change.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eExpression of the catechol-O-methyl transferase gene (\\u003cem\\u003eCOMT\\u003c/em\\u003e):\\u003c/strong\\u003e In the control group, the expression level was 26.410 with a ∆Ct of \\u0026ndash;13.520. In the treated groups, the expression levels at low, medium, and high doses were 23.765, 25.530, and 28.395, respectively.\\u0026nbsp;The housekeeping gene (HK) showed a stable expression level across all samples. The comparison of gene expression levels (∆ Ct)\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003erevealed a down-regulation in the treated groups compared to the control. Particularly, at low, medium, and high doses, the ∆ Ct values were -5.870, -6.610, and -6.330, respectively (Table 2).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe 2^-(∆∆ Ct)\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003evalues of the control are 1.000, serving as reference points for comparison. The corresponding 2^- (∆∆ Ct)\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003evalues at treated groups demonstrated a dose-dependent response.\\u0026nbsp;As the dose increased, there was a consistent reduction in COMT expression, with 2^\\u0026ndash;(∆∆Ct) values of 0.005, 0.008, and 0.007, respectively, compared to the control (Table 2 and Figure 2).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTable 2: Expression of catechol-O-methyl transferase gene (COMT)\\u003c/p\\u003e\\n\\u003ctable border=\\\"0\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"598\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd rowspan=\\\"2\\\" valign=\\\"bottom\\\" style=\\\"width: 189px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eSample\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"bottom\\\" style=\\\"width: 182px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eA-Ct\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd rowspan=\\\"2\\\" valign=\\\"bottom\\\" style=\\\"width: 68px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e∆ Ct\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd rowspan=\\\"2\\\" valign=\\\"bottom\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e∆∆ Ct\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd rowspan=\\\"2\\\" valign=\\\"bottom\\\" style=\\\"width: 92px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2^-(∆∆ ct)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 109px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eGOI(COMT)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 74px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eHK\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 189px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eControl\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 109px;\\\"\\u003e\\n \\u003cp\\u003e26.410\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 74px;\\\"\\u003e\\n \\u003cp\\u003e39.930\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 68px;\\\"\\u003e\\n \\u003cp\\u003e-13.520\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e0.000\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 92px;\\\"\\u003e\\n \\u003cp\\u003e1.000\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 189px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTreated at low dose\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 109px;\\\"\\u003e\\n \\u003cp\\u003e23.765\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 74px;\\\"\\u003e\\n \\u003cp\\u003e29.635\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 68px;\\\"\\u003e\\n \\u003cp\\u003e-5.870\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e7.650\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 92px;\\\"\\u003e\\n \\u003cp\\u003e0.005\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 189px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTreated at medium dose\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 109px;\\\"\\u003e\\n \\u003cp\\u003e25.530\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 74px;\\\"\\u003e\\n \\u003cp\\u003e32.140\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 68px;\\\"\\u003e\\n \\u003cp\\u003e-6.610\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e6.910\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 92px;\\\"\\u003e\\n \\u003cp\\u003e0.008\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 189px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTreated at high dose\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 109px;\\\"\\u003e\\n \\u003cp\\u003e28.395\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 74px;\\\"\\u003e\\n \\u003cp\\u003e34.725\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 68px;\\\"\\u003e\\n \\u003cp\\u003e-6.330\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 66px;\\\"\\u003e\\n \\u003cp\\u003e7.190\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 92px;\\\"\\u003e\\n \\u003cp\\u003e0.007\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003eKeys: A-Ct= Average Ct value, GOI= Gene of interest, HK= House Keeping Gene, \\u003cstrong\\u003e∆ Ct\\u003c/strong\\u003e = delta Ct value, \\u003cstrong\\u003e∆∆ Ct\\u003c/strong\\u003e= delta delta Ct value and \\u003cstrong\\u003e2^-(∆∆ ct) =\\u0026nbsp;\\u003c/strong\\u003eFold Change.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eExpression of mu-1 opioid receptor (OPRM1) gene\\u003c/strong\\u003e: the control group serves as the baseline for comparisons, with a Gene of interest (GOI) expression of (20.8750) and housekeeping gene expression of 39.9300. The ∆ Ct\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eis (-19.0550), indicating the baseline difference between the GOI and HK.\\u0026nbsp;\\u003cstrong\\u003eThe low-dose treated group showed a decrease in the expression of the gene of interest (GOI), with a significant ∆∆Ct value of 12.6300 and a corresponding 2^\\u0026ndash;(∆∆Ct) value of 0.0002, indicating notable\\u0026nbsp;\\u003c/strong\\u003edown-regulation\\u003cstrong\\u003e. Similarly, the medium- and high-dose groups both exhibited consistent and significant\\u0026nbsp;\\u003c/strong\\u003edown-regulation\\u003cstrong\\u003e, with identical ∆∆Ct values of 9.0850 and 2^\\u0026ndash;(∆∆Ct) values of 0.0018, compared to the control (Table 3 and Figure 3).\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTable 3: Expression of mu-1 opioid receptor (OPRM1)\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003egene\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"624\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd rowspan=\\\"2\\\" valign=\\\"bottom\\\" style=\\\"width: 178px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eSample\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"bottom\\\" style=\\\"width: 191px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eA-Ct\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd rowspan=\\\"2\\\" valign=\\\"bottom\\\" style=\\\"width: 91px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e∆ Ct\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd rowspan=\\\"2\\\" valign=\\\"bottom\\\" style=\\\"width: 75px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e∆∆ Ct\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd rowspan=\\\"2\\\" valign=\\\"bottom\\\" style=\\\"width: 89px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2^-(∆∆ ct)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 116px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eGOI(OPRM1)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 75px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;HK\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 178px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eControl\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 116px;\\\"\\u003e\\n \\u003cp\\u003e20.8750\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 75px;\\\"\\u003e\\n \\u003cp\\u003e39.9300\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 91px;\\\"\\u003e\\n \\u003cp\\u003e-19.0550\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 75px;\\\"\\u003e\\n \\u003cp\\u003e0.0000\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 89px;\\\"\\u003e\\n \\u003cp\\u003e1.0000\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 178px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTreated at low dose\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 116px;\\\"\\u003e\\n \\u003cp\\u003e23.2100\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 75px;\\\"\\u003e\\n \\u003cp\\u003e29.6350\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 91px;\\\"\\u003e\\n \\u003cp\\u003e-6.4250\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 75px;\\\"\\u003e\\n \\u003cp\\u003e12.6300\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 89px;\\\"\\u003e\\n \\u003cp\\u003e0.0002\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 178px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTreated at medium dose\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 116px;\\\"\\u003e\\n \\u003cp\\u003e22.1700\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 75px;\\\"\\u003e\\n \\u003cp\\u003e32.1400\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 91px;\\\"\\u003e\\n \\u003cp\\u003e-9.9700\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 75px;\\\"\\u003e\\n \\u003cp\\u003e9.0850\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 89px;\\\"\\u003e\\n \\u003cp\\u003e0.0018\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 178px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTreated at high dose\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 116px;\\\"\\u003e\\n \\u003cp\\u003e24.7550\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 75px;\\\"\\u003e\\n \\u003cp\\u003e34.7250\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 91px;\\\"\\u003e\\n \\u003cp\\u003e-9.9700\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 75px;\\\"\\u003e\\n \\u003cp\\u003e9.0850\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\" style=\\\"width: 89px;\\\"\\u003e\\n \\u003cp\\u003e0.0018\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003eKeys: A-Ct= Average Ct value, GOI= Gene of interest, HK= House Keeping Gene,\\u0026nbsp;\\u003cstrong\\u003e∆ Ct\\u003c/strong\\u003e = delta Ct value,\\u0026nbsp;\\u003cstrong\\u003e∆∆ Ct\\u003c/strong\\u003e= delta delta Ct value and\\u0026nbsp;\\u003cstrong\\u003e2^-(∆∆ ct) =\\u0026nbsp;\\u003c/strong\\u003eFold Change.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"DISCUSSION\",\"content\":\"\\u003cp\\u003eThe results of the present study confirm that sub-chronic (60-day) administration of varying doses of Fearless Energy Drink (FED) altered the expression levels of the dopamine D\\u003csub\\u003e2\\u003c/sub\\u003e receptor (\\u003cem\\u003eDRD\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e) in the hippocampal region of the brains of albino rats. The gene of interest (GOI), \\u003cem\\u003eDRD\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e, demonstrated different expression levels across different treatment groups. Remarkably, the treated groups display varying degrees of down-regulation compared to the control, as indicated by negative ∆ Ct values. Pharmacologically, the down-regulation of the \\u003cem\\u003eDRD\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e gene, which encodes dopamine D\\u003csub\\u003e2\\u003c/sub\\u003e receptors, may result in reduced receptor availability, potentially leading to an increase in dopamine release. This could result in elevated levels of dopamine in the synaptic cleft (Noble, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2000\\u003c/span\\u003e). This is in agreement with growing evidence from several studies that suggests caffeine intoxication from EDs, and that EDs may serve as a gateway to other forms of drug dependence (Griffiths \\u003cem\\u003eet al\\u003c/em\\u003e., 2019). Another study reported that coffee extract and its components, such as caffeine, may influence gene expression by altering histone modifications, which are essential for regulating the physical properties of chromatin and its corresponding transcriptional state, DNA methylation, and non-coding RNA (ncRNA) expression (Ding et al., \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Similarly, a separate study found that a psychostimulant such as caffeine induced down-regulation of \\u003cem\\u003eDRD\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e mRNA expression in the testes of rats (Gonzalez et al., \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). The gene expression study for catechol-O-methyl transferase (\\u003cem\\u003eCOMT\\u003c/em\\u003e) analysed the expression of the GOI, \\u003cem\\u003eCOMT\\u003c/em\\u003e, across different samples, including a control group and treated samples at low, medium, and high doses. The FED consumption revealed that the \\u003cem\\u003eCOMT\\u003c/em\\u003e gene exhibited differential expression levels across the experimental groups. This implies that the treatment influenced down-regulation the expression of the addictive gene \\u003cem\\u003eCOMT\\u003c/em\\u003e. As the dose increases, there is a consistent decrease in \\u003cem\\u003eCOMT\\u003c/em\\u003e expression. The \\u003cem\\u003eCOMT\\u003c/em\\u003e gene encodes catechol-O-methyltransferase, an enzyme involved in the breakdown of dopamine. The down-regulation of the \\u003cem\\u003eCOMT\\u003c/em\\u003e gene could lead to reduced activity of this enzyme, potentially resulting in decreased dopamine metabolism. As a result, there might be an accumulation of dopamine in the synaptic cleft, prolonging its presence and enhancing neurotransmission (Simpson et al., \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; Kennedy et al., \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). Caffeinated energy drinks may indirectly affect the \\u003cem\\u003eCOMT\\u003c/em\\u003e enzyme, potentially influencing dopamine levels. However, the relationship is complex, and the precise mechanisms remain incompletely understood.\\u003c/p\\u003e\\u003cp\\u003eIt was also noted that the gene expression study for mu-1 opioid receptor (\\u003cem\\u003eOPRM1\\u003c/em\\u003e) gene reveals alteration in the expression of the GOI: \\u003cem\\u003eOPRM1\\u003c/em\\u003e under different treatment samples compared to the control. The data include the expression of the gene of interest (\\u003cem\\u003eOPRM1\\u003c/em\\u003e), the housekeeping gene (HK), and the corresponding ∆Ct values. The 2^- (∆∆ Ct) (fold change value) proposes a significant down-regulation in gene expression, which emphasises the potential influence of the fearless energy drink on the regulation of the OPRM1 gene. The \\u003cem\\u003edown-regulation\\u003c/em\\u003e induced by FED in the present findings on the OPRM1 gene could potentially impact the opioid system, leading to alterations in pain perception, mood regulation, reward processing, and possibly vulnerability to conditions involving dysregulation of the opioid and dopamine systems, such as addiction (Picci et al., \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). The result of the current study agreed with some findings of the gene expression studies that caffeinated energy drinks induced alteration of gene expression in various organ of the body like that of El-Terras et al. (\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e), reported that carbonated soft drinks contained caffeine, were also observed to down-regulation the expression of monoaminoxidase-A (\\u003cem\\u003eMAO-A\\u003c/em\\u003e) and acetylcholinesterase (AchE) serum and mRNA level in the brain. Additionally, there was an up-regulation of mRNA expression of the dopamine D\\u003csub\\u003e2\\u003c/sub\\u003e receptor (\\u003cem\\u003eDRD\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e), while the expression of the 5-hydroxytryptamine transporter (\\u003cem\\u003e5HTT\\u003c/em\\u003e) was decreased (El-Terras et al., \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). Additional findings of the gene expression study reported that genes involved in neurogenesis were decreased, which could be related to the impairment of memory function. Ethanol significantly altered the expression of 416 out of 11,727 genes expressed in the ventral hippocampus. While in the medial prefrontal cortex, 638 out of 11,579 were altered; genes in cellular stress and inflammatory pathways were increased, as were genes involved in oxidative phosphorylation (McClintick et al., \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Caffeine is a central nervous system stimulant and can interact with various genes and neurotransmitter systems. However, its primary mode of action involves adenosine receptors rather than direct modulation of \\u003cem\\u003eDRD\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e, \\u003cem\\u003eCOMT\\u003c/em\\u003e, or \\u003cem\\u003eOPRM1\\u003c/em\\u003e genes.\\u003c/p\\u003e\\u003cp\\u003eThe dopamine neurotransmitter plays a crucial role in addiction by reinforcing behaviours associated with pleasure or reward. High levels of dopamine can contribute to the development of addiction by reinforcing the desire to repeat actions that lead to pleasure, such as drug use, alcohol intake or certain behaviours like gambling and heavy activities.\\u003c/p\\u003e\\u003cp\\u003eA 500 ml bottle of Fearless Energy Drink (FED) contains approximately 155 mg of caffeine, which is proposed to be responsible for the observed changes in gene expression levels, due to the high caffeine content of energy drinks (Al-Shaar et al., \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). However, the concentration of the caffeine content in the fearless energy drink singlehandedly cannot induced the observed changes (Al-Basher et al., \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e), but a combination of the caffeine and other active stimulants present in the energy drink like taurine, guarana and herbal extracts were proposed to potentiates the observed changes (Zeidan-Chulia et al., \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e).\\u003c/p\\u003e\"},{\"header\":\"CONCLUSION\",\"content\":\"\\u003cp\\u003eThe gene expression findings concluded that the three addictive genes of interest were dose-dependently down-regulated in response to FED, with higher doses exhibiting the most pronounced effects, resulting in a substantial reduction in the expression levels of the genes studied. However, the down-regulatory effect of FED was more pronounced on the \\u003cem\\u003eDRD2\\u003c/em\\u003e gene than on the \\u003cem\\u003eCOMT\\u003c/em\\u003e gene, and similarly, more pronounced on the \\u003cem\\u003eCOMT\\u003c/em\\u003e gene than on the \\u003cem\\u003eOPRM1\\u003c/em\\u003e gene.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eRecommendation\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003eBased on the observed dose-dependent down-regulation of the three addictive genes of interest following administration of Fearless Energy Drink, further investigations are recommended to:\\u003c/p\\u003e\\u003cp\\u003e\\u003cul\\u003e\\u003cli\\u003e\\u003cp\\u003eExplore the specific mechanisms underlying the observed alteration of the gene expression.\\u003c/p\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cp\\u003eValidate the results using additional experimental approaches or biological assays.\\u003c/p\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cp\\u003eValidate these findings using complementary techniques and possibly expand the study to include other relevant genes or factors that could contribute to the observed effects.\\u003c/p\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cp\\u003eCollaborate with experts in the field to gain insights into the broader context of the observed gene expression changes.\\u003c/p\\u003e\\u003c/li\\u003e\\u003c/ul\\u003e\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003eFED: Fearless energy drink; ED: Energy drink;\\u003cem\\u003e\\u0026nbsp;DRD\\u003csub\\u003e2\\u003c/sub\\u003e\\u003c/em\\u003e: Dopamine receptor D\\u003csub\\u003e2\\u003c/sub\\u003e subtype; SUD: Substance use disorder; PTSD: Post-traumatic stress disorder; \\u003cem\\u003eCOMT\\u003c/em\\u003e: catechol-O-methyltransferase; \\u003cem\\u003eOPRM1\\u003c/em\\u003e: Opioid receptor Miu-1; PBS: Phosphate-buffered saline; NFW: Nuclease free water; DMSO: Dimethyl Sulfoxide; NG-Tube: Nasogastric tube; AREC: Animal Research and Ethical Committee; DNA: Deoxyribonucleic acid; RNA: Ribonucleic acid; RPM: Rotation per minutes; PCR: Polymerase chain reaction; HK: Housekeeping gene; GOI: Gene of interest; A-Ct: Average Ct value;\\u0026nbsp;Ct: delta Ct value;\\u0026nbsp;\\u003cstrong\\u003e∆∆\\u0026nbsp;\\u003c/strong\\u003eCt: delta delta Ct value;\\u0026nbsp;2^-(∆∆ ct):\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eFold Change; \\u003cem\\u003eMAO-A\\u003c/em\\u003e: Monoaminoxidase-A; AchE: Acetylcholinesterase; CA1: Cornu ammonis-1; DG: Dentate gyrus.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgement\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thankfully acknowledged the contribution of Prof. Usman Sadiq, the Dean of the Faculty of Pharmacy, Unimaid, and Prof. Bala Usman Shamaki of Veterinary Pharmacology, Faculty of Veterinary Medicine. Unimaid, for their support, supervision, and guidance during this research.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contribution\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eYHAG, FSD, GUS, and TSY initiated the work, YHAG and FSD carried out the laboratory animal handling and treatment, while GUS supervised the work and TSY analysed the data. YHAG and FSD wrote the draft of the manuscript. GUS revised the draft, while all authors approved the final version of the work before submission to the journal.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNo financial support was received for this research.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe corresponding author will be provided with data upon request.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll experimental procedures were carried out in strict accordance with the Guide to the Care and Use of Laboratory Animals in Research and Teaching, as detailed in NIH publications (Council 2011).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no conflict of interest.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor details\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eDepartment of Pharmacology and Toxicology. Faculty of Pharmacy, University of Maiduguri, Maiduguri, Borno State, Nigeria.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eAdjene, J.O., Ezeoke, J.C., Nwose, E.U. (2010). Histological effects of chronic consumption of soda pop drinks on kidney of adult Wistar rats. \\u003cem\\u003eNorth America Journal of Medical Science,\\u003c/em\\u003e 2(1): 215‑217. \\u003c/li\\u003e\\n\\u003cli\\u003eAkande, I.S. and Banjoko, O.A. (2011). Assessment of biochemical effect of \\u0026ldquo;power horse\\u0026rdquo; energy drink on hepatic, renal and histological functions in sprague dawley rats. \\u003cem\\u003eAnnual review \\u0026amp; research in biology\\u003c/em\\u003e, 1(3), 45-56. \\u003c/li\\u003e\\n\\u003cli\\u003eAl-Basher, G.I., Aljabal, H., Almeer, R., Allam, A.A. and Mahmoud, A.M. (2018). Perinatal exposure to energy drink induces oxidative damage in the liver, kidney and brain, and behavioral alterations in mice offspring. \\u003cem\\u003eBiomedical Pharmacotherapy\\u003c/em\\u003e, 102: 798-811. \\u003c/li\\u003e\\n\\u003cli\\u003eAl-Shaar, L., Vercammen, K., Lu, C., Richard- Son, S., Tamez, M. And Mattei, J. (2017). Health effects and public health concerns of energy drink consumption in the United States: A Mini-Review. \\u003cem\\u003eFront. Public health\\u003c/em\\u003e, 5: 225. \\u003c/li\\u003e\\n\\u003cli\\u003eAmerican Addiction Centre, (2023). Is drug addiction genetic? Retrieved 4\\u003csup\\u003eth\\u003c/sup\\u003e January, 2024 from \\u003cem\\u003e. \\u003c/em\\u003e\\u003c/li\\u003e\\n\\u003cli\\u003eBano, S.S., Shabana, A., Saddaf, A., Ali, A., and Imran, B. (2020). Effects of caffeinated energy drink withdrawal on effects of caffeinated energy drink withdrawal on histological and biochemical parameters of adult histological and biochemical parameters of adult albino rat kidneys. \\u003cem\\u003eJournal of Medicinal Science\\u003c/em\\u003e, 28(2), 107-111. \\u003c/li\\u003e\\n\\u003cli\\u003eCadoni, C. and Peana, A.T. (2023). Energy drinks at adolescences: awareness or unawareness. \\u003cem\\u003eFrontiers in Behavioural Neuroscience\\u003c/em\\u003e, 2023: 1-8. \\u003c/li\\u003e\\n\\u003cli\\u003eCharles river, (n.d) Sub-chronic and chronic toxicity studies. Retrieved on 4\\u003csup\\u003eth\\u003c/sup\\u003e February, 2024, from \\u003c/li\\u003e\\n\\u003cli\\u003eDing, Q., Xu, Y., and Lau, A.T.Y. (2023). The epigenetic effects of coffee. \\u003cem\\u003eJournal Molecules\\u003c/em\\u003e, 28(4), 1770. \\u003c/li\\u003e\\n\\u003cli\\u003eEl-Terras, A., Mohammed, M.S., Alkhedaide, A., Fouad, A.H., Alharthy, A., and Elah, A.B. (2016). Carbonated soft drinks induced oxidative stress and altered expression of certain genes in the brain of wistar rats. \\u003cem\\u003eMolecular Medicine Reports\\u003c/em\\u003e, 13(4), 3147-3154. \\u003c/li\\u003e\\n\\u003cli\\u003eErdmann, J., Wiciński, M., W\\u0026oacute;dkiewicz, E., Nowaczewska, M., Słupski, M., Otto, S.W., Kubiak, K., Huk-Wieliczuk, E. and Malinowski, B. (2021). Effects of Energy Drink Consumption on Physical Performance and Potential Danger of Inordinate Usage. \\u003cem\\u003eNutrients\\u003c/em\\u003e, 13: 2506. . \\u003c/li\\u003e\\n\\u003cli\\u003eFerreira, E.S., Hartmann Quadros, M.I., Trindade, A.A., Takahashi, S., Koyama, G.R., and Souza-Formigoni, O.M. (2004). Can energy drinks reduce the depressor effect of ethanol? An experimental study in mice. \\u003cem\\u003ePhysiology and Behaviour\\u003c/em\\u003e, 82(5), 841-847. \\u003c/li\\u003e\\n\\u003cli\\u003eGenetic Science Leaning Centre, (2020). Genes and addiction. Retrieved January 02, 2024, from . \\u003c/li\\u003e\\n\\u003cli\\u003eGonzalez, C.R., Gonzalez, B., Matzkin, M.E., Muniz, J.A., Cadet, J.L., Garcia-Rill, E. (2015). Psychostimulant induced testicular toxicity in mice; evidence of cocaine and caffeine on the local dopaminergic system. \\u003cem\\u003ePlos One\\u003c/em\\u003e, 10(11), 0142713. \\u003c/li\\u003e\\n\\u003cli\\u003eGriffiths, R.R., Chad, J.R., Erick, C.S. (2009). Caffeinated energy drink. A growing problem. \\u003cem\\u003eJournal of Drug and Alcohol Dependence\\u003c/em\\u003e, 99(3), 1-10. \\u003c/li\\u003e\\n\\u003cli\\u003eJesupemi, A. (2023). Alert: NAFDAC warns against consumption of energy drink brand. \\u003cem\\u003eTheCable\\u003c/em\\u003e, (November 3, 2023), retrieved from https//www.thecable.ng on January 5, 2024. \\u003c/li\\u003e\\n\\u003cli\\u003eJordan, C.J and Xi, X (2022). Identification of the risk genes associated with vulnerability to addiction: major findings from transgenic animals. Front. Neurosci., 15 (2021). \\u003c/li\\u003e\\n\\u003cli\\u003eKennedy, J.L., Arqam, Q., Clement, C.Z., Yuko, H., Arun, K.T., Sheraz, C., Behdin, N., and Joseph, H.B. (2015). The role of the catechol-o-methyltransferase (COMT) gene val158met in aggressive behaviour, a review of genetic studies. \\u003cem\\u003eCurrent Neuropharmacology\\u003c/em\\u003e, 13, 802-814. \\u003c/li\\u003e\\n\\u003cli\\u003eMansy, W., Deema, M.A., Mona, H., Enas, Z. (2017). Effects of chronic consumption of energy drinks on liver and kidney of experimental rats. \\u003cem\\u003eTropical Journal of Pharmaceutical Research\\u003c/em\\u003e, 16 (12): 2849-2856. \\u003c/li\\u003e\\n\\u003cli\\u003eMcClintick, J.N., McBride, W.J., Bell, R.L., Dina, Z., and Liu, Y. (2018). Gene expression changes in the ventral hippocampus and medial prefrontal cortex of adolescent alcohol-preferring (p) rats following binge-like alcohol drinking. \\u003cem\\u003eJournal of Alcohol\\u003c/em\\u003e, 68, 37-47. \\u003c/li\\u003e\\n\\u003cli\\u003eMuxiddinovna, I.M. (2022). Effects of energy drinks on biochemical and sperm parameters in albino rats. \\u003cem\\u003eCentral Asian Journal of Medical and Natural Sciences\\u003c/em\\u003e, 3(3): 2660-4159. \\u003c/li\\u003e\\n\\u003cli\\u003eNational Institute on Drug Abuse, NIDA. (2020). Drugs, brain, and behaviour: the science of addiction drug misuse and addiction. Retrieved September 5\\u003csup\\u003eth\\u003c/sup\\u003e 2023 from . \\u003c/li\\u003e\\n\\u003cli\\u003eNoble, P.E (2000). The DRD2 in psychiatric and neurological disorders and its phenotypes. \\u003cem\\u003ePharmacogenomics\\u003c/em\\u003e, 1(3), 309-333. \\u003c/li\\u003e\\n\\u003cli\\u003eOlatona, F.A., Ijeoma, O.A., Sunday, A.A., and Temitope, W.L.A. (2018). Energy drinks consumption among football players in Lagos, Nigeria. \\u003cem\\u003eSouth African Journal of Clinical Nutrition\\u003c/em\\u003e, 31(4), 84\\u0026ndash;88. \\u003c/li\\u003e\\n\\u003cli\\u003ePicci, G., Fishbein, D.H., Vanmeter, J.W., Rose, E.J. (2022). Effects of OPRM1 and DRD2 on brain structure in drug-na\\u0026iuml;ve adolescents: genetic and neural vulnerabilities to substance use. \\u003cem\\u003ePsychopharmacology (Berl)\\u003c/em\\u003e, 239(1): 141-152. \\u003c/li\\u003e\\n\\u003cli\\u003eSimpson, E.H., Morud, J., Winiger, V., Biezonski, D., Zhu, J.P., Bach, M.E., Malleret, G., Polan, H.J., Ng-Evan, S., Phillip, P.E.M., Kellendonk, C. and Kandel, E.R. (2014). Genetic variation in COMT activity impacts learning and dopamine release capacity in the striatum. \\u003cem\\u003eLearning and Memory\\u003c/em\\u003e, 21(4), 205-214. \\u003c/li\\u003e\\n\\u003cli\\u003eSobel, A. (2025). How many energy drinks a day is safe?. . Published online on 24\\u003csup\\u003eth\\u003c/sup\\u003e June, 2025. Retrieved online on 5\\u003csup\\u003eth\\u003c/sup\\u003e July, 2025. \\u003c/li\\u003e\\n\\u003cli\\u003eTabassum, Y., Maheen, H., Khan, B., Zafar, I.B., Qamar, J., Abdul, R. and Muhammad, U. (2021). Constructive effects of energy drink consumption on players\\u0026rsquo; performance. \\u003cem\\u003ePalarch`s Journal of Archaeology Egypt/Egyptology\\u003c/em\\u003e, 18(8), 4099-4107. \\u003c/li\\u003e\\n\\u003cli\\u003eVargiu, R., Francesca, B., Carla, L., Daniele, L., Alessandro, C., Pier, P.B. and Valentina, B. (2021). Chronic red bull consumption during adolescence: Effect on mesocortical and mesolimbic dopamine transmission and cardiovascular system in adult rats. , 2021, 14, 609. \\u003c/li\\u003e\\n\\u003cli\\u003eZeidan-Chulia, F., Gelain, D.P., Kolling, E.A., Rybarczyk-Filho, J.L., Ambrosi, P., Resende, T.S. (2013). Major components of energy drinks (caffeine, taurine, and guarana) exert cytotoxic effects on human neuronal SH-SY5Y cells by decreasing reactive oxygen species production. \\u003cem\\u003eOxid. Med. Cell\\u003c/em\\u003e. ID791795. \\u003c/li\\u003e\\n\\u003cli\\u003eZhou, F., Maren, A.N., Pawel, Z., Liao, Y.K., Lu, H., Duduit, R.J., Huang, D., Ashrafi, H., Zhao, T., Hueeta, I.A., Ranney, G.T., and Liu, W. (2021). An optimize protocol for stepwise optimization of real-Time RT-PCR analysis. \\u003cem\\u003eHorticulture Research\\u003c/em\\u003e, 8, 179. \\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Energy drink, Gene expression, Albino rats, down-regulation, Addictive genes\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7134304/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7134304/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e\\u003cp\\u003eAddiction to energy drinks (ED) is a growing concern, with potential impacts on neurobiology and gene expression in the hippocampal region. This study examines the impact of Fearless energy drink (FED) on the expression of addiction-related genes within the hippocampal region of albino rats following sub-chronic exposure. Twenty-four (24) adult albino rats were acclimatised and were randomly assigned into four groups: A, B, C, and D (n\\u0026thinsp;=\\u0026thinsp;6). Group A served as the control group, whereas Groups B, C, and D received oral administration of Fearless energy drink via oropharyngeal gavage at daily doses of 7 ml/kg, 14 ml/kg, and 21 ml/kg, respectively, for a continuous period of sixty days. At the end of the administration, brain tissue was excised for molecular studies, and gene expression was analysed using quantitative real-time polymerase chain reaction (qRT-PCR). The data were analysed following the manufacturer's instructions for the Real-time PCR instrument.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e\\u003cp\\u003eThe gene expression analysis focused on three genes of interest: The dopamine D\\u003csub\\u003e2\\u003c/sub\\u003e receptor (DRD\\u003csub\\u003e2\\u003c/sub\\u003e), catechol-O-methyl transferase (COMT), and Mu-1 opioid receptor (OPRM1), all of which are associated with addictive behaviour. These target genes exhibited differential expression levels across the various treatment groups. Notably, the treated samples exhibit varying degrees of down-regulation compared to the control, as indicated by negative delta Ct (∆ Ct\\u003cb\\u003e)\\u003c/b\\u003e values. This study suggests that the treatment had an impact on suppressing the expression levels of the addictive genes, highlighting the potential impact of the fearless Energy drink on the regulation of the addictive genes of interest.\\u003c/p\\u003e\\u003ch2\\u003eConclusion\\u003c/h2\\u003e\\u003cp\\u003e\\u003cem\\u003eThe study demonstrates that\\u003c/em\\u003e Fearless \\u003cem\\u003eenergy drink causes a dose-dependent suppression of addiction-related genes (DRD\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003e2\\u003c/em\\u003e\\u003c/sub\\u003e, \\u003cem\\u003eCOMT, and OPRM1) in the hippocampus of albino rats. These findings suggest that chronic consumption of energy drinks may disrupt normal neurobiological gene regulation and could contribute to addiction-related changes in the brain.\\u003c/em\\u003e\\u003c/p\\u003e\",\"manuscriptTitle\":\"Gene Expression Study of Some Addictive Genes in the Hippocampal Region of Albino Rats Treated with a Brand of Energy Drink at Sub-Chronic Period\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-08-07 17:47:42\",\"doi\":\"10.21203/rs.3.rs-7134304/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"e819bfb2-7a35-4916-81ae-c1f0659db029\",\"owner\":[],\"postedDate\":\"August 7th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-09-29T08:23:39+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-08-07 17:47:42\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7134304\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7134304\",\"identity\":\"rs-7134304\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}