Effects of E-Cigarette Exposure with/out CDP-Choline Treatment on Withdrawal-Induced Anxiety, and Hormonal Levels: Using Rat Model

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Alzaghari, Mohammad A.A. Al-Najjar, Thanaa Al-zuhd, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7184123/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract E-cig use is increasingly popular, yet its impact on mental health, particularly anxiety and hormonal regulation during withdrawal, is understudied. Cytidine 5'-diphosphocholine (CDP-choline), a neuroprotective compound, may help manage withdrawal symptoms. This study explores the effects of CDP-choline on withdrawal-induced anxiety and hormonal imbalances from chronic e-cig exposure, focusing on serum levels of nicotine, cotinine, adrenaline, and beta-endorphins. Male Wistar rats were divided into five groups: control, e-cig-exposed, e-cigarette-exposed with CDP-choline, e-cigarette quitting with CDP-choline, and CDP-choline-only. E-cig exposure involved one hour, twice daily, five days a week for six weeks, followed by reduced exposure for three weeks. CDP-choline was administered for three weeks starting at week six. Behavioral tests, including the light and dark box (LDB) test, were conducted at baseline, during withdrawal, and post-treatment. E-cig exposure significantly elevated serum nicotine, cotinine, adrenaline, and beta-endorphin levels, while increasing anxiety-like behaviors. CDP-choline treatment effectively reduced nicotine and cotinine levels, particularly in the e-cig exposure + CDP group (p = 0.0027) and the quitting + CDP group (p = 0.0416). Additionally, CDP-choline substantially lowered adrenaline and beta-endorphin levels (p < 0.0001), reducing stress responses linked to withdrawal. In conclusion, CDP-choline mitigates the harmful effects of e-cig exposure by reducing hormonal imbalances and improving behavioral outcomes. These findings highlight its potential as a therapeutic option for managing e-cig-induced withdrawal symptoms. Health sciences/Diseases Health sciences/Medical research Biological sciences/Neuroscience e-cig exposure anxiety-like behavior addiction CDP-choline hormonal levels neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Electronic cigarettes (e-cigs) are nicotine delivery devices, that utilize a battery to heat and aerosolize e-liquid, which is held in a refillable reservoir, producing vapor for the user to inhale [ 1 – 3 ]. The liquid often consists of propylene glycol, vegetable glycerin, and different flavoring ingredients in addition to nicotine [ 4 ]. E-cigs are available in a variety of nicotine strengths ranging from 0 to 36 mg/mL, as well as a variety of flavors designed to appeal to kids and teens [ 5 ]. In the past ten years, e-cigs have become more readily available, with an estimated 58.1 million users in 2018 and 68 million in 2020 globally [ 6 ]. Furthermore, e-cigs are frequently marketed as safer alternatives to conventional cigarettes and even as a tool for quitting smoking since they are expected to have lesser amounts of toxins than traditional tobacco products [ 7 ]. Unfortunately, the US Food and Drug Administration (FDA) appears to lack standards for e-cigs as therapeutic medicine delivery systems and plans to regulate them as tobacco products [ 8 ]. Several studies have indicated that e-cigs produce a visible and inhalable aerosol, not just nicotine vapor, as wrongly claimed by e-cig companies. E-cig aerosols contain measurable amounts of toxins, such as formaldehyde, benzene, acetaldehyde, acrolein, phenols, metals, and reactive oxygen species [ 2 , 9 , 10 ]. Additionally, e-cig usage can cause damage by inhaling the stimulant substance nicotine, which can create dependency and increase exposure to the e-cig aerosol [ 11 ]. This leads to continual inhalation of toxins that can be generated when propylene glycol and vegetable glycerin are heated [ 5 , 12 , 13 ]. Previous studies have shown that e-cig exposure can impair development and produce an allergy-based asthma inflammatory response in mice [ 14 , 15 ]. E-cigs can also increase oxidative stress and inflammation in mice and decrease immunological responses against bacterial and viral infections [ 16 ]. Moreover, several studies have shown that nicotine addiction causes cognitive and working memory impairments, mood disorders such as stress, and anxiety-like symptoms [ 17 , 18 ]. Consistent with the above, results from animal experiments suggest that chronic exposure to nicotine via minipumps, intravenous self-administration, or e-cigs results in a typical dependence marked by a withdrawal syndrome, which includes somatic symptoms, anxiety, and anhedonia, as indicated by an elevated reward threshold [ 19 – 21 ]. A recent study has shown the impact of e-cigs on the adrenal gland cortex, which concluded that e-cig exposure developed histopathological changes in the adrenal cortex structure, with significant improvement in its histological structure and biochemical activity following discontinuation [ 22 ]. Smoking also impacts pituitary, thyroid, adrenal, testicular, calcium metabolism, and insulin action in our body [ 23 , 24 ]. Furthermore, a prior study demonstrated that nicotine interacts with receptors in the adrenal medulla, resulting in a rise in blood pressure, accelerated respiratory rate, a faster heart rate, and raised levels of glucose in the blood due to enhanced release of adrenaline and noradrenaline [ 25 ]. Cytidine-5-phosphocholine (CDP-choline) is an intermediate in phosphatidylcholine production, a necessary component of cellular membranes, and a cell signaling mediator [ 26 ]. It is delivered orally, intravenously, or intracerebroventricularly and converted to choline and cytidine/uridine via the Kennedy pathway [ 27 ], resulting in elevated plasma and tissue concentrations of these metabolites [ 28 ]. CDP-choline has been used to treat traumatic brain injury [ 29 ] and cerebral ischemia [ 30 ], showing positive results, great tolerance, and few adverse effects [ 31 , 32 ]. Its protective impact has been linked to membrane stability by suppressing phospholipase A activation, maintaining sphingomyelin levels, and boosting phosphatidylcholine [ 33 , 34 ]. An initial investigation discovered that CDP-choline reduces oxidative stress-induced apoptosis in cardiomyocytes exposed to hypoxia [ 35 ]. Although prior research showed that CDP-choline therapy is effective in a variety of neurodegenerative diseases [ 36 ], also it can reduce inflammation via the peripheral cholinergic system by mediating α7 nicotine acetylcholine receptors on macrophages [ 28 ]. Thus, in the present study, we investigate the effect of chronic e-cig exposure and CDP-choline treatment on different hormonal levels including nicotine, cotinine, adrenalin, and beta-endorphins. Furthermore, the effect of e-cig exposure, as well as CDP-choline treatment on anxiety-like behavior was examined. Materials and Methods Drugs VOOPOO DRAG M100S e-cig, 3 mg/ml Nasty mango juice, CITICOLIN (250 mg Cytidine − 5’-diphosphocholine, Cognizin®, Kyowa Hakko Bio Co., Ltd.). Animal management Thirty-five male adult Wistar rats weighing (180–200 g) were inbred at Applied Science Private University in Jordan. Animals were acclimated for 1 week in a vivarium room, kept on a (12-h/12-h light/dark cycle), and maintained under a controlled temperature (23 ± 2°C) and humidity (50 ± 5%). All animal experiments were approved by the Research and Ethics Committee of the Faculty of Pharmacy at Applied Science Private University, Amman, Jordan (Approval No.: 2023-PHA-26). All procedures were performed in accordance with institutional guidelines and national regulations for the care and use of laboratory animals. This study is reported in accordance with the ARRIVE guidelines 2.0 to ensure rigorous and transparent reporting of animal research. Twenty-four hours before the experiment, rats went through a behavioral test to have baseline behavioral data. A Canon digital camera was used to record the videos. The behavioral test was manually assessed by an observer who was blind to the experiment. The behavioral test was used to measure anxiety-like behavior using the dark and light box test (DLB). After obtaining the behavior results, rats were divided into five groups with a similar average baseline in behavior and weight. For biochemical and behavioral testing, the rats were divided into 5 groups (n = 7): control group (C), group exposed to e-cig smoke (E-cig), group exposed to e-cig smoke + CDP-choline treatment (E-cig + CDP), group that quit the e-cig and given CDP-choline treatment (E-cig quitting + CDP), and group given CDP-choline treatment only (CDP), as shown on Fig. 1 A. The group of control rats served as the untreated group. Rat weight evaluation Rats were weighed three times for each phase at the baseline, after the exposure, and after the treatment phase to evaluate changes occurring through the experiment. Behavioral tests In the DLB test, the apparatus consists of two chambers one of which is lighted (40×40×40 cm) and the other is darkened (20×40×40 cm) with an entrance between them (7.5×8.5 cm). Rats are introduced into the light compartment, where they investigate the surrounding area until they discover the entrance to the dark chamber. The light chamber was illuminated by a neon tube on the ceiling, generating a brightness of 150 lux in the center of the box [ 37 ]. Each rat was placed in the apparatus for 10 minutes at each session [ 38 ], and the time spent in the light chamber, latency to enter the dark chamber, and frequency of stretching were recorded. All chambers are cleansed with water after each session. E-cig exposure setting The rats were exposed to the vapor of an e-cig for 9 weeks using a custom-designed acrylic exposure chamber with a dimension of 50×50×50 cm, for 1 hour twice daily with a 3-hour resting period of exposure, as shown in Fig. 1 B. VOOPOO DRAG M100S e-cigs (Coil: PnP-TW20 (0.2Ω), Glass Container: Standard (5 ml capacity), UK) with a nicotine level of 3 mg/mL (Mango Flavor; Nasty, Malaysia) were used. For 2 hours of nicotine exposure, rats were exposed to two VOOPOO e-cig cartridges for a total nicotine exposure of 27 mg (9 ml × 3 mg/mL). Five-second puffs with twenty-second inter-puff intervals were utilized during the exposure. After 6 weeks of e-cig exposure, the exposure time was minimized to an hour per day, and the treatment phase was initiated. Serum collection Two ml of blood were collected in a tube using retro-orbital bleeding [ 39 ]. After blood collection, the blood stayed for 45 minutes at room temperature before centrifugation. The centrifuge process continued for 15 minutes at 5500 rpm to separate the serum. The blood samples were collected three times including the baseline, after the exposure, and after the treatment phase. Quantitation of serum nicotine, cotinine, adrenalin, and β-endorphin via Ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) Nicotine and cotinine This study used a validated SHIMADZU UFLC-MS/MS system (Waters Corporation, Milford, MA, USA) to determine the concentration of nicotine and cotinine in rat serum. The chromatographic procedures involved the use of a C 18 column (50 × 4.6 mm, 0.5 µm) with 75% acetonitrile and 0.05% formic acid as mobile phase in an isocratic elution at a flow rate of 0.3 ml/min in a total run time of 4 minutes. The internal standard used in this experiment was methanol. The eluted compounds were detected using LCMS-8030As LIQUID CHROMATOGRAPH MASS SPECTROMETER-Triple Quad MS (Waters Corporation, Milford, MA, USA) supplied with an electrospray ionization source (ESI) running in positive ionization mode to generate [M + H] + ion at m/z 162.23, 176.21 for nicotine and cotinine, respectively. The parameters were optimized as follows: source temperature 120°C, desolvation temperature 300°C, desolvation gas flow 600 L/h, and cone gas flow 60 L/h. The capillary voltage was set at 3 kV, while the cone voltage was set at 15 V and high-purity nitrogen was used during the MS operations. Adrenaline The same UFLC-MS/MS system was carried out to determine the concentration of adrenaline in rat serum. The chromatographic procedures involved the use of a C 18 column (100 × 2.1 mm, 1.7 µm) with a binary mobile phase mixture of methanol and water (50:50, v/v) at a flow rate of 0.3 ml/minute in a total run time of 3.8 minutes. The eluted compounds were detected using the same MS-TQ system supplied with an ESI source running in positive ionization mode to generate [M + H] + ion at m/z 184 for adrenalin. β-endorphins UFLC-MS/MS system was carried out to determine the concentration of β-endorphins in rat serum. The chromatographic procedures involved the use of a C 18 column (100 mm × 1.8 mm, 1.7 µm) with a binary mobile phase mixture of methanol and water (55:45, v/v) at a flow rate of 0.25 ml/minute in a total run time of 4.2 minutes. The eluted compounds were detected using an MS-TQ system supplied with an ESI source running in positive ionization mode to generate [M + H] + ion at m/z 3529 for β-endorphins. Statistical analysis Data were compiled as means and standard errors of the means (SEM). Two-way repeated measures ANOVA, followed by One-way ANOVA as well as Tukey multiple comparisons, were used to analyze data for weight, and behavioral tests. One-way ANOVA followed by Tukey’s multiple comparisons was used to investigate the hormonal levels. All results were statistically analyzed using GraphPad Prism 9 (GraphPad Software, Inc., San Diego, CA, United States). Differences were considered to be statistically significant at probability values of P equal or less than 0.05. Results Effect of e-cig whole body exposure and CDP treatment on rats' weights Absolute body weight and gains for rats in each group at different time points are shown in Fig. 2 . This pattern of effects was confirmed by two-way repeated measures ANOVA revealing a significant main effect of Time [F (1.82, 54.49) = 165.8, p < 0.0001], while the main effect of Treatment [F (4, 30) = 1.19, ns], and the Time x Treatment interaction [F (8, 60) = 1.36, ns] were not significant. Tukey’s multiple comparisons revealed significant increases in body weight after the exposure phase compared to the baseline. In addition, there was a significant increase in body weight after 21 days of treatment compared to after the exposure phase for all groups (*p < 0.05, **p < 0.01; Fig. 2 ). Effect of e-cig exposure and CDP treatment on DLB test E-cig exposure caused withdrawal-induced anxiety in the DLB test that developed after 6 weeks of e-cig exposure, seen as a decrease in the time spent in the light chamber, as demonstrated in Fig. 3 A. This effect was reversed by treatment with CDP. This pattern of effects was confirmed by repeated measures of two-way ANOVA, which revealed a significant main effect of Time [F (1.71, 51.30) = 9.32, p = 0.0007], a significant main effect of Treatment [F (4, 30) = 3.84, p = 0.012] and a significant Time × Treatment interaction [F (8, 60) = 3.38, p = 0.0029]. Tukey multiple comparison tests revealed a significant decrease in time spent in the light chamber in E-cig, E-cig + CDP, and E-cig quitting + CDP groups compared to control and CDP groups after the exposure phase. This effect was reversed after 21-days of CDP treatment. There was a significant difference in time spent in the light chamber in the e-cig group compared to the control, and CDP groups, but there was no difference between E-cig + CDP, and E-cig quitting + CDP groups compared to control and CDP groups. A similar pattern was observed for latency to enter the dark chamber. Repeated measures of two-way ANOVA revealed a significant main effect of Time [F (1.47, 43.94) = 6.54, p = 0.0069] and a significant main effect of Treatment [F (4, 30) = 6.43, p = 0.0007] and a significant Time × Treatment interaction [F (8, 60) = 2.36, p = 0.028]. Tukey multiple comparison tests revealed a significant decrease in latency to enter the dark chamber in E-cig, E-cig + CDP, and E-cig quitting + CDP groups compared to control and CDP groups after the exposure phase. There was a significant decrease in latency to enter the dark chamber in the e-cig group compared to E-cig quitting + CDP, control, and CDP groups after 21-days of CDP treatment, as shown in Fig. 3 B. In the same manner, the frequency of stretching was increased after e-cig exposure in the E-cig, E-cig + CDP, and E-cig quitting + CDP groups compared to the control and CDP groups. Repeated measures of two-way ANOVA revealed a significant main effect of Time [F (1.71, 51.39) = 10.55, p = 0.0003] and a significant main effect of Treatment [F (4, 30) = 9.02, p < 0.0001] and a significant Time × Treatment interaction [F (8, 60) = 5.39, p < 0.0001]. Tukey multiple comparison tests revealed a significant increase in the frequency of stretching in E-cig, E-cig + CDP, and E-cig quitting + CDP groups compared to control and CDP groups after the exposure phase. There was a significant increase in the frequency of stretching in the e-cig group compared to E-cig quitting + CDP, control, and CDP groups after 21-days of CDP treatment, as shown in Fig. 3 C. Effect of e-cig exposure and CDP treatment on serum nicotine, cotinine, adrenaline, and β-endorphins concentrations The analysis of nicotine concentrations in rat serum following exposure to e-cig vaping with and without CDP-cytidine treatment reveals significant differences. When compared to the negative control, nicotine levels were significantly higher in both the 2-hour and 1-hour e-cig exposure groups, with mean differences of -8.250 and − 5.100, respectively, indicating strong significance (p < 0.0001 and p = 0.0002). However, the addition of CDP-cytidine to the 1-hour e-cig group reduced the nicotine concentration, showing no significant difference compared to the control (mean diff. -1.250, p = 0.1955), as shown in Fig. 4 A. Similarly, e-cig quitting combined with CDP-cytidine treatment did not significantly differ from the control (mean diff. -1.090, p = 0.2883). When comparing different e-cig exposure durations, the 2-hour exposure had substantially higher nicotine concentrations than the 1-hour group (mean diff. 3.150, p = 0.0035), with CDP-cytidine reducing nicotine levels in both cases. Nicotine levels in the CDP-cytidine alone group showed no difference from the control (p > 0.9999). Additionally, comparisons between e-cig exposure treated CDP-cytidine-treated groups confirmed a substantial nicotine reduction, particularly between the 2-hour exposure and the CDP-treated groups (p < 0.0001). Figure 4 B illustrates cotinine concentrations in rat serum following exposure to e-cig aerosol with and without CDP-cytidine treatment and shows significant variations across different conditions. Cotinine levels were markedly higher in the 2-hour and 1-hour e-cig exposure groups compared to the negative control, with mean differences of -633.7 and − 343.0, respectively, showing strong statistical significance (p < 0.0001). In contrast, the addition of CDP-cytidine to the 1-hour e-cig exposure group significantly reduced cotinine levels (mean diff. -242.7, p < 0.0001), though still higher than the control. The e-cig quitting group combined with CDP-cytidine showed a further reduction in cotinine levels, with a smaller yet significant difference from the control (mean diff. -122.7, p = 0.0167). Cotinine levels in the CDP-cytidine alone group showed no significant difference from the control (p > 0.9999). When comparing the e-cig exposure groups, the 2-hour exposure showed significantly higher cotinine levels than the 1-hour group (mean diff. 290.7, p < 0.0001). Similarly, cotinine levels were significantly lower in the 1-hour e-cig group when CDP-cytidine was introduced (mean diff. 100.3, p = 0.0287). The most substantial reduction in cotinine levels was observed between the 2-hour e-cig group and the quitting group with CDP-cytidine (mean diff. 511.0, p < 0.0001); highlighting CDP-cytidine’s potential to reduce cotinine concentrations. Moreover, significant differences were noted when comparing the 1-hour e-cig exposure group to its CDP-treated counterparts (mean diff. 220.3, p < 0.0001). Adrenaline levels were notably higher in the 2-hour and 1-hour e-cig exposure groups compared to the control (p < 0.0001), as illustrated in Fig. 4 C. CDP-cytidine treatment significantly reduced adrenaline levels in both the 1-hour e-cig (p = 0.0027) and e-cig quitting groups (p = 0.0416), while CDP-cytidine alone showed no effect. The 2-hour e-cig group had significantly higher levels than the 1-hour group (p = 0.0068), but CDP-cytidine treatment further reduced levels (p = 0.0143). The most significant reduction occurred in the 2-hour exposure group with CDP treatment (p < 0.0001), highlighting CDP-cytidine's role in lowering adrenaline levels across different exposure durations. In addition, beta-endorphin levels in rat serum after e-cig exposure, with and without CDP-cytidine, show significant results, as shown in Fig. 4 D. Both the 2-hour and 1-hour e-cig exposure groups had markedly higher beta-endorphin levels compared to the control (p < 0.0001 and p = 0.0001, respectively). CDP-cytidine treatment reduced levels in the 1-hour exposure group, but the difference was not statistically significant (p = 0.0893), nor was it significant in the e-cig quitting group (p = 0.7377). The 2-hour e-cig group had significantly higher levels than the 1-hour group (p = 0.0015), and CDP treatment further lowered beta-endorphin levels across all groups (p < 0.0001), with the largest reduction seen in the 2-hour exposure group. However, no significant differences were found between the CDP-treated and quitting groups. Discussion E-cig exposure and CDP treatment were examined for the first time on withdrawal-induced anxiety, inflammation, and serum hormone levels. This study shows that e-cig exposure alters nicotine, cotinine, adrenaline, and beta-endorphin levels in rats, affecting hormonal balance and behavior. These data also suggest that CDP-choline (cytidine) may reduce the adverse effects of e-cigs. Chronic exposure to e-cig in this study resulted in significantly elevated serum levels of nicotine, cotinine, adrenaline, and beta-endorphin. Nicotine and its primary metabolite, cotinine, are well-known to interfere with the normal functioning of the endocrine system by triggering the release of catecholamines such as adrenaline. This elevation in catecholamines, particularly adrenaline, leads to heightened stress responses and is consistent with previous studies linking nicotine exposure to increased oxidative stress and neuroinflammation [ 40 , 41 ]. Beta-endorphin, a hormone linked to the body’s pain and stress response systems, was also significantly increased following e-cig exposure, particularly in the 2-hour group. This finding aligns with reports that nicotine can stimulate the release of endogenous opioids like beta-endorphins, which contribute to nicotine's reinforcing effects and addictive properties [ 42 ]. The elevated beta-endorphin levels observed in this study suggest that e-cig exposure may enhance the rewarding and addictive potential of nicotine, creating a cycle that reinforces dependency and exacerbates hormonal dysregulation in exposed animals [ 40 ]. E-cig-exposed rats' nicotine, cotinine, adrenaline, and beta-endorphin levels were significantly reduced by CDP-choline for 3 weeks, especially in the 1-hour exposure and quitting groups. CDP-choline reduces these effects by restoring cell membrane phospholipid metabolism, increasing neurotransmitter production (especially acetylcholine), and improving neural plasticity [ 43 ]. In this study, CDP-choline treatment notably reduced anxiety-like behaviors, as evidenced by the LDB results. These behavioral improvements coincide with reductions in neuroinflammation and oxidative stress [ 44 ]. CDP-choline also had a pronounced effect on the reduction of adrenaline and beta-endorphin levels, particularly in the quitting + CDP group. This suggests that CDP-choline may aid in the normalization of stress hormone levels post-exposure, which is critical for recovery from nicotine addiction and withdrawal symptoms [ 45 ]. The lower levels of these hormones observed in the CDP-treated groups likely contributed to the reduction in anxiety behavior, as excessive levels of adrenaline and beta-endorphins are associated with stress and mood disorders [ 46 , 47 ]. The control and CDP-treated groups had similar beta-endorphin levels, demonstrating the compound's potential to restore hormonal balance without causing abnormal hormonal reactions. This reinforces research indicating that CDP-choline reduces external stresses and maintains endocrine system homeostasis [ 48 ]. Multifaceted processes explain CDP-choline's protective properties. It is commonly known that CDP-choline is a precursor of phosphatidylcholine, a significant cell membrane component. This helps maintain membrane fluidity, release neurotransmitters, and repair neurons [ 49 ]. These findings have significant implications for the development of therapeutic strategies aimed at mitigating the harmful effects of e-cigs. CDP-choline’s ability to alleviate both biochemical and behavioral changes induced by e-cig exposure underscores its potential as a therapeutic agent for nicotine addiction and its associated neurological and hormonal disturbances. Future studies should explore its long-term efficacy and potential use in clinical settings for treating e-cig addiction and neurotoxicity in humans. Conclusion In summary, this study provides strong evidence that chronic e-cig exposure leads to significant hormonal disruptions and neurobehavioral changes, including increased anxiety, and stress hormone levels. CDP-choline treatment was effective in reversing these effects by reducing serum nicotine, cotinine, adrenaline, and beta-endorphin levels, as well as improving behavioral outcomes. Declarations Ethical approval and ARRIVE compliance All animal experiments were approved by the Research and Ethics Committee of the Faculty of Pharmacy at Applied Science Private University, Amman, Jordan (Approval No.: 2023-PHA-26). All procedures were performed in accordance with institutional guidelines and national regulations for the care and use of laboratory animals. This study is reported in accordance with the ARRIVE guidelines 2.0 to ensure rigorous and transparent reporting of animal research. Consent for publication Not applicable. The manuscript does not contain any individual data or identifiable personal information. Competing interests The authors declare that they have no competing interests. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors’ contributions M.B. and A.K. conceptualized and designed the study. D.R. and S.A. contributed to data collection and initial drafting. M.B. and W.A. performed data analysis and interpretation. A.K., S.A., and M.A.S. contributed to literature review and manuscript editing. M.B. supervised the project and finalized the manuscript. All authors read and approved the final version. Acknowledgment The authors would like to thank the Applied Science Private University, Jordan for supporting this work. Data availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. References Unger, M. and D.W. Unger, E-cigarettes/electronic nicotine delivery systems: a word of caution on health and new product development. Journal of thoracic disease, 2018. 10 (Suppl 22): p. S2588. 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Zafonte, R., et al., The citicoline brain injury treatment (COBRIT) trial: design and methods. Journal of neurotrauma, 2009. 26 (12): p. 2207-2216. Ortega, G., et al., Citicoline treatment prevents neurocognitive decline after a first ischemic stroke. Cerebrovasc Dis, 2010. 29 (Suppl 2): p. 268-288. Saver, J.L., Citicoline: update on a promising and widely available agent for neuroprotection and neurorepair. Rev Neurol Dis, 2008. 5 (4): p. 167-177. Cho, H.-J. and Y.J. Kim, Efficacy and safety of oral citicoline in acute ischemic stroke: drug surveillance study in 4,191 cases. Methods and findings in experimental and clinical pharmacology, 2009. 31 (3): p. 171-176. Lee, J.M., et al., A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects. Nature, 2011. 474 (7352): p. 506-510. Liu, Y., et al., Eicosapentaenoic acid-enriched phosphatidylcholine attenuated hepatic steatosis through regulation of cholesterol metabolism in rats with nonalcoholic fatty liver disease. Lipids, 2017. 52 : p. 119-127. Hernández-Esquivel, L., et al., Cardioprotective properties of citicoline against hyperthyroidism-induced reperfusion damage in rat hearts. Biochemistry and Cell Biology, 2015. 93 (3): p. 185-191. Mir, C., et al., CDP-choline prevents glutamate-mediated cell death in cerebellar granule neurons. Journal of Molecular Neuroscience, 2003. 20 : p. 53-59. Griebel, G., G. Perrault, and D.J. Sanger, Characterization of the behavioral profile of the non-peptide CRF receptor antagonist CP-154,526 in anxiety models in rodents Comparison with diazepam and buspirone: Comparison with diazepam and buspirone. Psychopharmacology, 1998. 138 : p. 55-66. Belovicova, K., et al., Animal tests for anxiety-like and depression-like behavior in rats. Interdisciplinary toxicology, 2017. 10 (1): p. 40. Sharma, A., et al., Safety and blood sample volume and quality of a refined retro-orbital bleeding technique in rats using a lateral approach. Lab animal, 2014. 43 (2): p. 63-66. Picciotto, M.R. and P.J. Kenny, Mechanisms of nicotine addiction. Cold Spring Harbor perspectives in medicine, 2021. 11 (5): p. a039610. Chatterjee, S., et al., Acute exposure to e-cigarettes causes inflammation and pulmonary endothelial oxidative stress in nonsmoking, healthy young subjects. American Journal of Physiology-Lung Cellular and Molecular Physiology, 2019. 317 (2): p. L155-L166. Zalewska-Kaszubska, J., Beta-endorphin peptide and some selected psychiatric disorders. Journal of Psychiatry Studies, 2018. 1 (1). Secades, J.J. and G. Frontera, CDP-choline: pharmacological and clinical review. Methods and findings in experimental and clinical pharmacology, 1995. 17 : p. 1-54. Xu, M., et al., Choline deficiency and choline metabolism disorders can lead to anxiety-like behaviors induced by DSS in IBD mice. 2022. Torres, O.V., et al., Nicotine withdrawal increases stress-associated genes in the nucleus accumbens of female rats in a hormone-dependent manner. Nicotine & Tobacco Research, 2015. 17 (4): p. 422-430. Merenlender-Wagner, A., Y. Dikshtein, and G. Yadid, The β-endorphin role in stress-related psychiatric disorders. Current drug targets, 2009. 10 (11): p. 1096-1108. Imam-Fulani, A. and B.V. Owoyele, Effect Of Caffeine and Adrenaline on Memory and Anxiety in Male Wistar Rats. Nigerian Journal of Physiological Sciences, 2022. 37 (1): p. 69-76. Secades, J., Citicoline in the treatment of cognitive impairment. J Neurol Exp Neurosci, 2019. 5 (1): p. 14-26. Arenth, P.M., et al., CDP-choline as a biological supplement during neurorecovery: a focused review. PM&R, 2011. 3 (6): p. S123-S131. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 28 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 26 Aug, 2025 Reviews received at journal 24 Aug, 2025 Reviews received at journal 24 Aug, 2025 Reviewers agreed at journal 17 Aug, 2025 Reviewers agreed at journal 14 Aug, 2025 Reviewers agreed at journal 14 Aug, 2025 Reviewers invited by journal 13 Aug, 2025 Editor assigned by journal 13 Aug, 2025 Editor invited by journal 31 Jul, 2025 Submission checks completed at journal 24 Jul, 2025 First submitted to journal 24 Jul, 2025 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. 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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-7184123","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":501920019,"identity":"dd4f833c-0379-473b-aa4b-09023fc611b6","order_by":0,"name":"Muna Barakat","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYFACHhCRwMDA3gDmMjaAecRo4eE5QLIWiQS4FvxAt4H34OeKmjR5e8nHjz/zMNjIbjjA8PABPi1mB/iSJc8cyzHskU4zk+ZhSDMGakk2wK+Fx0Cyga2CsUc6wYyZh+FwIlBLmgQBLcY/G/5V2PdIHv8MdNh/orSYSTa25ST2SPAYAB12gBgtfGmWjX1pyT1ncsok5xgkG888TNAvvIdvNnxLtm1vP775w5sKO9m+4z2JD/BpYZBHkQYZz8yTgFcHNsB+gGQto2AUjIJRMKwBAJxhSTIV3BalAAAAAElFTkSuQmCC","orcid":"","institution":"Applied Science Private University","correspondingAuthor":true,"prefix":"","firstName":"Muna","middleName":"","lastName":"Barakat","suffix":""},{"id":501920021,"identity":"d4d6f4af-5714-4637-8362-af2faf3a8951","order_by":1,"name":"Lujain F. Alzaghari","email":"","orcid":"","institution":"Applied Science Private University","correspondingAuthor":false,"prefix":"","firstName":"Lujain","middleName":"F.","lastName":"Alzaghari","suffix":""},{"id":501920023,"identity":"ab92e968-233d-4c51-9b2e-5aba82cc444c","order_by":2,"name":"Mohammad A.A. Al-Najjar","email":"","orcid":"","institution":"Applied Science Private University","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"A.A.","lastName":"Al-Najjar","suffix":""},{"id":501920024,"identity":"6b156b73-20e6-4a82-8e2c-6ff360c805b0","order_by":3,"name":"Thanaa Al-zuhd","email":"","orcid":"","institution":"Applied Science Private University","correspondingAuthor":false,"prefix":"","firstName":"Thanaa","middleName":"","lastName":"Al-zuhd","suffix":""},{"id":501920027,"identity":"7779b7d8-9117-421b-8417-8f46b4ef79a0","order_by":4,"name":"Maram Abdaljaleel","email":"","orcid":"","institution":"The University of Jordan","correspondingAuthor":false,"prefix":"","firstName":"Maram","middleName":"","lastName":"Abdaljaleel","suffix":""},{"id":501920028,"identity":"44a48c2d-3892-41f1-a8d6-59dded7a5be7","order_by":5,"name":"Luay Abu-Qatouseh","email":"","orcid":"","institution":"University of Petra","correspondingAuthor":false,"prefix":"","firstName":"Luay","middleName":"","lastName":"Abu-Qatouseh","suffix":""},{"id":501920031,"identity":"b6b92cdd-8fb3-4454-bf78-de655cb604ca","order_by":6,"name":"Samar Thiab","email":"","orcid":"","institution":"Applied Science Private University","correspondingAuthor":false,"prefix":"","firstName":"Samar","middleName":"","lastName":"Thiab","suffix":""},{"id":501920035,"identity":"614ce556-bd12-4f6f-ab0c-72d4fa4b0a88","order_by":7,"name":"Abdelrahim Alqudah","email":"","orcid":"","institution":"The Hashemite University","correspondingAuthor":false,"prefix":"","firstName":"Abdelrahim","middleName":"","lastName":"Alqudah","suffix":""}],"badges":[],"createdAt":"2025-07-22 07:53:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7184123/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7184123/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-26799-z","type":"published","date":"2025-11-28T15:57:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89490370,"identity":"8fe42d56-0aff-4d14-9e2b-a18ee14b3965","added_by":"auto","created_at":"2025-08-20 13:44:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1131126,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Experimental timeline for e-cig exposure, CDP treatment, behavioral testing, and serum collection; (B) Diagram illustrating the apparatus used for e-cig exposure. This figure has been created using Biorender® scientific illustration software\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7184123/v1/1686a169f50d33aac02507a5.png"},{"id":89490379,"identity":"8f11449d-de0d-4316-89bc-2b8a38cef392","added_by":"auto","created_at":"2025-08-20 13:44:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":131297,"visible":true,"origin":"","legend":"\u003cp\u003eAbsolute body weight and gains for E-cig, E-cig + CDP, E-cig quitting + CDP, control, and CDP groups at different time points (mean ± SEM). Two-way ANOVA repeated measure revealed significant increases in body weight after the exposure phase compared to the baseline. In addition, there was a significant increase in body weight after 21-days of treatment compared to after the exposure phase for all groups (*p \u0026lt;0.05, **p \u0026lt; 0.01) (n=7 for each group)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7184123/v1/c7053dc2464d34fae2836165.png"},{"id":89490378,"identity":"9ee3e3fb-a95d-4596-b548-a4b4fb524367","added_by":"auto","created_at":"2025-08-20 13:44:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":720185,"visible":true,"origin":"","legend":"\u003cp\u003eBehavioral testing in the DLB test (mean ± SEM). (A) Time spent in the light chamber of the DLB inE-cig, E-cig + CDP, E-cig quitting + CDP, control, and CDP groups at different time points; (B) Latency to enter the dark chamber of the DLB in E-cig, E-cig + CDP, E-cig quitting + CDP, control, and CDP groups at different time points (C) Frequency of stretching of the DLB in E-cig, E-cig + CDP, E-cig quitting + CDP, control, and CDP groups at different time points (*p \u0026lt; 0.05, **p \u0026lt; 0.01, n = 7 for each group).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7184123/v1/9f4889436113e42c06270bae.png"},{"id":89490373,"identity":"96d34a6e-385c-43ee-b36f-49bcfb68aa45","added_by":"auto","created_at":"2025-08-20 13:44:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":424200,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent hormonal serum concentrations post one- and two-hour E-cig exposure with/out cytidine. (A) Nicotine serum concertation shows significant differences between one and two hours were presented as ** for p-value \u0026lt; 0.01. A significant difference between the cytidine-treated rats with and without 1-hour E-cig exposure presented as ## for p-value \u0026lt; 0.01 and ### for p-value \u0026lt; 0.001; (B) Cotinine serum concentration shows significant differences between one and two hours were presented as ** for p-value \u0026lt; 0.01. Significant differences between the cytidine-treated rats with and without 1-hour E-cig exposure presented as # for p-value \u0026lt; 0.05 and ### for p-value \u0026lt; 0.001; (C) Adrenaline serum concentration shows significant differences between one and two hours were presented as ** for p-value \u0026lt; 0.01. Significant differences between the cytidine-treated rats with and without 1-hour E-cig exposure presented as # for p-value \u0026lt; 0.05 and ### for p-value \u0026lt; 0.001; (D) Beta-endorphins shows significant differences between one and two hours were presented as ** for p-value \u0026lt; 0.01. Significant differences between the cytidine-treated rats with and without One-hour E-cig exposure presented as # for p-value \u0026lt; 0.05 and ## for p-value \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7184123/v1/50c409fd6426bf17538bb903.png"},{"id":97179332,"identity":"238b4d10-8f2c-4bb2-bd83-267bf01d7aca","added_by":"auto","created_at":"2025-12-01 16:14:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3405429,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7184123/v1/2e29c7fc-8ea9-4fd6-8a31-748cb13811b0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of E-Cigarette Exposure with/out CDP-Choline Treatment on Withdrawal-Induced Anxiety, and Hormonal Levels: Using Rat Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eElectronic cigarettes (e-cigs) are nicotine delivery devices, that utilize a battery to heat and aerosolize e-liquid, which is held in a refillable reservoir, producing vapor for the user to inhale [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The liquid often consists of propylene glycol, vegetable glycerin, and different flavoring ingredients in addition to nicotine [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. E-cigs are available in a variety of nicotine strengths ranging from 0 to 36 mg/mL, as well as a variety of flavors designed to appeal to kids and teens [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In the past ten years, e-cigs have become more readily available, with an estimated 58.1\u0026nbsp;million users in 2018 and 68\u0026nbsp;million in 2020 globally [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Furthermore, e-cigs are frequently marketed as safer alternatives to conventional cigarettes and even as a tool for quitting smoking since they are expected to have lesser amounts of toxins than traditional tobacco products [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Unfortunately, the US Food and Drug Administration (FDA) appears to lack standards for e-cigs as therapeutic medicine delivery systems and plans to regulate them as tobacco products [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSeveral studies have indicated that e-cigs produce a visible and inhalable aerosol, not just nicotine vapor, as wrongly claimed by e-cig companies. E-cig aerosols contain measurable amounts of toxins, such as formaldehyde, benzene, acetaldehyde, acrolein, phenols, metals, and reactive oxygen species [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Additionally, e-cig usage can cause damage by inhaling the stimulant substance nicotine, which can create dependency and increase exposure to the e-cig aerosol [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This leads to continual inhalation of toxins that can be generated when propylene glycol and vegetable glycerin are heated [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrevious studies have shown that e-cig exposure can impair development and produce an allergy-based asthma inflammatory response in mice [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. E-cigs can also increase oxidative stress and inflammation in mice and decrease immunological responses against bacterial and viral infections [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Moreover, several studies have shown that nicotine addiction causes cognitive and working memory impairments, mood disorders such as stress, and anxiety-like symptoms [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Consistent with the above, results from animal experiments suggest that chronic exposure to nicotine via minipumps, intravenous self-administration, or e-cigs results in a typical dependence marked by a withdrawal syndrome, which includes somatic symptoms, anxiety, and anhedonia, as indicated by an elevated reward threshold [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA recent study has shown the impact of e-cigs on the adrenal gland cortex, which concluded that e-cig exposure developed histopathological changes in the adrenal cortex structure, with significant improvement in its histological structure and biochemical activity following discontinuation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Smoking also impacts pituitary, thyroid, adrenal, testicular, calcium metabolism, and insulin action in our body [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, a prior study demonstrated that nicotine interacts with receptors in the adrenal medulla, resulting in a rise in blood pressure, accelerated respiratory rate, a faster heart rate, and raised levels of glucose in the blood due to enhanced release of adrenaline and noradrenaline [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCytidine-5-phosphocholine (CDP-choline) is an intermediate in phosphatidylcholine production, a necessary component of cellular membranes, and a cell signaling mediator [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. It is delivered orally, intravenously, or intracerebroventricularly and converted to choline and cytidine/uridine via the Kennedy pathway [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], resulting in elevated plasma and tissue concentrations of these metabolites [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. CDP-choline has been used to treat traumatic brain injury [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and cerebral ischemia [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], showing positive results, great tolerance, and few adverse effects [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Its protective impact has been linked to membrane stability by suppressing phospholipase A activation, maintaining sphingomyelin levels, and boosting phosphatidylcholine [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. An initial investigation discovered that CDP-choline reduces oxidative stress-induced apoptosis in cardiomyocytes exposed to hypoxia [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Although prior research showed that CDP-choline therapy is effective in a variety of neurodegenerative diseases [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], also it can reduce inflammation via the peripheral cholinergic system by mediating α7 nicotine acetylcholine receptors on macrophages [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThus, in the present study, we investigate the effect of chronic e-cig exposure and CDP-choline treatment on different hormonal levels including nicotine, cotinine, adrenalin, and beta-endorphins. Furthermore, the effect of e-cig exposure, as well as CDP-choline treatment on anxiety-like behavior was examined.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eDrugs\u003c/p\u003e\u003cp\u003eVOOPOO DRAG M100S e-cig, 3 mg/ml Nasty mango juice, CITICOLIN (250 mg Cytidine \u0026minus;\u0026thinsp;5\u0026rsquo;-diphosphocholine, Cognizin\u0026reg;, Kyowa Hakko Bio Co., Ltd.).\u003c/p\u003e\u003cp\u003eAnimal management\u003c/p\u003e\u003cp\u003eThirty-five male adult Wistar rats weighing (180\u0026ndash;200 g) were inbred at Applied Science Private University in Jordan. Animals were acclimated for 1 week in a vivarium room, kept on a (12-h/12-h light/dark cycle), and maintained under a controlled temperature (23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and humidity (50\u0026thinsp;\u0026plusmn;\u0026thinsp;5%). All animal experiments were approved by the Research and Ethics Committee of the Faculty of Pharmacy at Applied Science Private University, Amman, Jordan (Approval No.: 2023-PHA-26). All procedures were performed in accordance with institutional guidelines and national regulations for the care and use of laboratory animals. This study is reported in accordance with the ARRIVE guidelines 2.0 to ensure rigorous and transparent reporting of animal research.\u003c/p\u003e\u003cp\u003eTwenty-four hours before the experiment, rats went through a behavioral test to have baseline behavioral data. A Canon digital camera was used to record the videos. The behavioral test was manually assessed by an observer who was blind to the experiment. The behavioral test was used to measure anxiety-like behavior using the dark and light box test (DLB). After obtaining the behavior results, rats were divided into five groups with a similar average baseline in behavior and weight.\u003c/p\u003e\u003cp\u003eFor biochemical and behavioral testing, the rats were divided into 5 groups (n\u0026thinsp;=\u0026thinsp;7): control group (C), group exposed to e-cig smoke (E-cig), group exposed to e-cig smoke\u0026thinsp;+\u0026thinsp;CDP-choline treatment (E-cig\u0026thinsp;+\u0026thinsp;CDP), group that quit the e-cig and given CDP-choline treatment (E-cig quitting\u0026thinsp;+\u0026thinsp;CDP), and group given CDP-choline treatment only (CDP), as shown on Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The group of control rats served as the untreated group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRat weight evaluation\u003c/p\u003e\u003cp\u003eRats were weighed three times for each phase at the baseline, after the exposure, and after the treatment phase to evaluate changes occurring through the experiment.\u003c/p\u003e\u003cp\u003eBehavioral tests\u003c/p\u003e\u003cp\u003eIn the DLB test, the apparatus consists of two chambers one of which is lighted (40\u0026times;40\u0026times;40 cm) and the other is darkened (20\u0026times;40\u0026times;40 cm) with an entrance between them (7.5\u0026times;8.5 cm). Rats are introduced into the light compartment, where they investigate the surrounding area until they discover the entrance to the dark chamber. The light chamber was illuminated by a neon tube on the ceiling, generating a brightness of 150 lux in the center of the box [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Each rat was placed in the apparatus for 10 minutes at each session [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and the time spent in the light chamber, latency to enter the dark chamber, and frequency of stretching were recorded. All chambers are cleansed with water after each session.\u003c/p\u003e\u003cp\u003eE-cig exposure setting\u003c/p\u003e\u003cp\u003eThe rats were exposed to the vapor of an e-cig for 9 weeks using a custom-designed acrylic exposure chamber with a dimension of 50\u0026times;50\u0026times;50 cm, for 1 hour twice daily with a 3-hour resting period of exposure, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. VOOPOO DRAG M100S e-cigs (Coil: PnP-TW20 (0.2Ω), Glass Container: Standard (5 ml capacity), UK) with a nicotine level of 3 mg/mL (Mango Flavor; Nasty, Malaysia) were used. For 2 hours of nicotine exposure, rats were exposed to two VOOPOO e-cig cartridges for a total nicotine exposure of 27 mg (9 ml \u0026times; 3 mg/mL). Five-second puffs with twenty-second inter-puff intervals were utilized during the exposure. After 6 weeks of e-cig exposure, the exposure time was minimized to an hour per day, and the treatment phase was initiated.\u003c/p\u003e\u003cp\u003eSerum collection\u003c/p\u003e\u003cp\u003eTwo ml of blood were collected in a tube using retro-orbital bleeding [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. After blood collection, the blood stayed for 45 minutes at room temperature before centrifugation. The centrifuge process continued for 15 minutes at 5500 rpm to separate the serum. The blood samples were collected three times including the baseline, after the exposure, and after the treatment phase.\u003c/p\u003e\u003cp\u003eQuantitation of serum nicotine, cotinine, adrenalin, and β-endorphin via Ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS)\u003c/p\u003e\u003cp\u003eNicotine and cotinine\u003c/p\u003e\u003cp\u003eThis study used a validated SHIMADZU UFLC-MS/MS system (Waters Corporation, Milford, MA, USA) to determine the concentration of nicotine and cotinine in rat serum. The chromatographic procedures involved the use of a C\u003csub\u003e18\u003c/sub\u003e column (50 \u0026times; 4.6 mm, 0.5 \u0026micro;m) with 75% acetonitrile and 0.05% formic acid as mobile phase in an isocratic elution at a flow rate of 0.3 ml/min in a total run time of 4 minutes. The internal standard used in this experiment was methanol. The eluted compounds were detected using LCMS-8030As LIQUID CHROMATOGRAPH MASS SPECTROMETER-Triple Quad MS (Waters Corporation, Milford, MA, USA) supplied with an electrospray ionization source (ESI) running in positive ionization mode to generate [M\u0026thinsp;+\u0026thinsp;H]\u0026thinsp;+\u0026thinsp;ion at m/z 162.23, 176.21 for nicotine and cotinine, respectively. The parameters were optimized as follows: source temperature 120\u0026deg;C, desolvation temperature 300\u0026deg;C, desolvation gas flow 600 L/h, and cone gas flow 60 L/h. The capillary voltage was set at 3 kV, while the cone voltage was set at 15 V and high-purity nitrogen was used during the MS operations.\u003c/p\u003e\u003cp\u003eAdrenaline\u003c/p\u003e\u003cp\u003eThe same UFLC-MS/MS system was carried out to determine the concentration of adrenaline in rat serum. The chromatographic procedures involved the use of a C\u003csub\u003e18\u003c/sub\u003e column (100 \u0026times; 2.1 mm, 1.7 \u0026micro;m) with a binary mobile phase mixture of methanol and water (50:50, v/v) at a flow rate of 0.3 ml/minute in a total run time of 3.8 minutes. The eluted compounds were detected using the same MS-TQ system supplied with an ESI source running in positive ionization mode to generate [M\u0026thinsp;+\u0026thinsp;H]\u0026thinsp;+\u0026thinsp;ion at m/z 184 for adrenalin.\u003c/p\u003e\u003cp\u003eβ-endorphins\u003c/p\u003e\u003cp\u003eUFLC-MS/MS system was carried out to determine the concentration of β-endorphins in rat serum. The chromatographic procedures involved the use of a C\u003csub\u003e18\u003c/sub\u003e column (100 mm \u0026times; 1.8 mm, 1.7 \u0026micro;m) with a binary mobile phase mixture of methanol and water (55:45, v/v) at a flow rate of 0.25 ml/minute in a total run time of 4.2 minutes. The eluted compounds were detected using an MS-TQ system supplied with an ESI source running in positive ionization mode to generate [M\u0026thinsp;+\u0026thinsp;H]\u0026thinsp;+\u0026thinsp;ion at m/z 3529 for β-endorphins.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were compiled as means and standard errors of the means (SEM). Two-way repeated measures ANOVA, followed by One-way ANOVA as well as Tukey multiple comparisons, were used to analyze data for weight, and behavioral tests. One-way ANOVA followed by Tukey\u0026rsquo;s multiple comparisons was used to investigate the hormonal levels. All results were statistically analyzed using GraphPad Prism 9 (GraphPad Software, Inc., San Diego, CA, United States). Differences were considered to be statistically significant at probability values of P equal or less than 0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eEffect of e-cig whole body exposure and CDP treatment on rats' weights\u003c/p\u003e\u003cp\u003eAbsolute body weight and gains for rats in each group at different time points are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. This pattern of effects was confirmed by two-way repeated measures ANOVA revealing a significant main effect of Time [F (1.82, 54.49)\u0026thinsp;=\u0026thinsp;165.8, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001], while the main effect of Treatment [F (4, 30)\u0026thinsp;=\u0026thinsp;1.19, ns], and the Time x Treatment interaction [F (8, 60)\u0026thinsp;=\u0026thinsp;1.36, ns] were not significant. Tukey\u0026rsquo;s multiple comparisons revealed significant increases in body weight after the exposure phase compared to the baseline. In addition, there was a significant increase in body weight after 21 days of treatment compared to after the exposure phase for all groups (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eEffect of e-cig exposure and CDP treatment on DLB test\u003c/p\u003e\u003cp\u003eE-cig exposure caused withdrawal-induced anxiety in the DLB test that developed after 6 weeks of e-cig exposure, seen as a decrease in the time spent in the light chamber, as demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. This effect was reversed by treatment with CDP. This pattern of effects was confirmed by repeated measures of two-way ANOVA, which revealed a significant main effect of Time [F (1.71, 51.30)\u0026thinsp;=\u0026thinsp;9.32, p\u0026thinsp;=\u0026thinsp;0.0007], a significant main effect of Treatment [F (4, 30)\u0026thinsp;=\u0026thinsp;3.84, p\u0026thinsp;=\u0026thinsp;0.012] and a significant Time \u0026times; Treatment interaction [F (8, 60)\u0026thinsp;=\u0026thinsp;3.38, p\u0026thinsp;=\u0026thinsp;0.0029]. Tukey multiple comparison tests revealed a significant decrease in time spent in the light chamber in E-cig, E-cig\u0026thinsp;+\u0026thinsp;CDP, and E-cig quitting\u0026thinsp;+\u0026thinsp;CDP groups compared to control and CDP groups after the exposure phase. This effect was reversed after 21-days of CDP treatment. There was a significant difference in time spent in the light chamber in the e-cig group compared to the control, and CDP groups, but there was no difference between E-cig\u0026thinsp;+\u0026thinsp;CDP, and E-cig quitting\u0026thinsp;+\u0026thinsp;CDP groups compared to control and CDP groups. A similar pattern was observed for latency to enter the dark chamber. Repeated measures of two-way ANOVA revealed a significant main effect of Time [F (1.47, 43.94)\u0026thinsp;=\u0026thinsp;6.54, p\u0026thinsp;=\u0026thinsp;0.0069] and a significant main effect of Treatment [F (4, 30)\u0026thinsp;=\u0026thinsp;6.43, p\u0026thinsp;=\u0026thinsp;0.0007] and a significant Time \u0026times; Treatment interaction [F (8, 60)\u0026thinsp;=\u0026thinsp;2.36, p\u0026thinsp;=\u0026thinsp;0.028]. Tukey multiple comparison tests revealed a significant decrease in latency to enter the dark chamber in E-cig, E-cig\u0026thinsp;+\u0026thinsp;CDP, and E-cig quitting\u0026thinsp;+\u0026thinsp;CDP groups compared to control and CDP groups after the exposure phase. There was a significant decrease in latency to enter the dark chamber in the e-cig group compared to E-cig quitting\u0026thinsp;+\u0026thinsp;CDP, control, and CDP groups after 21-days of CDP treatment, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB. In the same manner, the frequency of stretching was increased after e-cig exposure in the E-cig, E-cig\u0026thinsp;+\u0026thinsp;CDP, and E-cig quitting\u0026thinsp;+\u0026thinsp;CDP groups compared to the control and CDP groups. Repeated measures of two-way ANOVA revealed a significant main effect of Time [F (1.71, 51.39)\u0026thinsp;=\u0026thinsp;10.55, p\u0026thinsp;=\u0026thinsp;0.0003] and a significant main effect of Treatment [F (4, 30)\u0026thinsp;=\u0026thinsp;9.02, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] and a significant Time \u0026times; Treatment interaction [F (8, 60)\u0026thinsp;=\u0026thinsp;5.39, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001]. Tukey multiple comparison tests revealed a significant increase in the frequency of stretching in E-cig, E-cig\u0026thinsp;+\u0026thinsp;CDP, and E-cig quitting\u0026thinsp;+\u0026thinsp;CDP groups compared to control and CDP groups after the exposure phase. There was a significant increase in the frequency of stretching in the e-cig group compared to E-cig quitting\u0026thinsp;+\u0026thinsp;CDP, control, and CDP groups after 21-days of CDP treatment, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eEffect of e-cig exposure and CDP treatment on serum nicotine, cotinine, adrenaline, and β-endorphins concentrations\u003c/p\u003e\u003cp\u003eThe analysis of nicotine concentrations in rat serum following exposure to e-cig vaping with and without CDP-cytidine treatment reveals significant differences. When compared to the negative control, nicotine levels were significantly higher in both the 2-hour and 1-hour e-cig exposure groups, with mean differences of -8.250 and \u0026minus;\u0026thinsp;5.100, respectively, indicating strong significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and p\u0026thinsp;=\u0026thinsp;0.0002). However, the addition of CDP-cytidine to the 1-hour e-cig group reduced the nicotine concentration, showing no significant difference compared to the control (mean diff. -1.250, p\u0026thinsp;=\u0026thinsp;0.1955), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. Similarly, e-cig quitting combined with CDP-cytidine treatment did not significantly differ from the control (mean diff. -1.090, p\u0026thinsp;=\u0026thinsp;0.2883). When comparing different e-cig exposure durations, the 2-hour exposure had substantially higher nicotine concentrations than the 1-hour group (mean diff. 3.150, p\u0026thinsp;=\u0026thinsp;0.0035), with CDP-cytidine reducing nicotine levels in both cases. Nicotine levels in the CDP-cytidine alone group showed no difference from the control (p\u0026thinsp;\u0026gt;\u0026thinsp;0.9999). Additionally, comparisons between e-cig exposure treated CDP-cytidine-treated groups confirmed a substantial nicotine reduction, particularly between the 2-hour exposure and the CDP-treated groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB illustrates cotinine concentrations in rat serum following exposure to e-cig aerosol with and without CDP-cytidine treatment and shows significant variations across different conditions. Cotinine levels were markedly higher in the 2-hour and 1-hour e-cig exposure groups compared to the negative control, with mean differences of -633.7 and \u0026minus;\u0026thinsp;343.0, respectively, showing strong statistical significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In contrast, the addition of CDP-cytidine to the 1-hour e-cig exposure group significantly reduced cotinine levels (mean diff. -242.7, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), though still higher than the control. The e-cig quitting group combined with CDP-cytidine showed a further reduction in cotinine levels, with a smaller yet significant difference from the control (mean diff. -122.7, p\u0026thinsp;=\u0026thinsp;0.0167). Cotinine levels in the CDP-cytidine alone group showed no significant difference from the control (p\u0026thinsp;\u0026gt;\u0026thinsp;0.9999). When comparing the e-cig exposure groups, the 2-hour exposure showed significantly higher cotinine levels than the 1-hour group (mean diff. 290.7, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Similarly, cotinine levels were significantly lower in the 1-hour e-cig group when CDP-cytidine was introduced (mean diff. 100.3, p\u0026thinsp;=\u0026thinsp;0.0287). The most substantial reduction in cotinine levels was observed between the 2-hour e-cig group and the quitting group with CDP-cytidine (mean diff. 511.0, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001); highlighting CDP-cytidine\u0026rsquo;s potential to reduce cotinine concentrations. Moreover, significant differences were noted when comparing the 1-hour e-cig exposure group to its CDP-treated counterparts (mean diff. 220.3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cp\u003eAdrenaline levels were notably higher in the 2-hour and 1-hour e-cig exposure groups compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC. CDP-cytidine treatment significantly reduced adrenaline levels in both the 1-hour e-cig (p\u0026thinsp;=\u0026thinsp;0.0027) and e-cig quitting groups (p\u0026thinsp;=\u0026thinsp;0.0416), while CDP-cytidine alone showed no effect. The 2-hour e-cig group had significantly higher levels than the 1-hour group (p\u0026thinsp;=\u0026thinsp;0.0068), but CDP-cytidine treatment further reduced levels (p\u0026thinsp;=\u0026thinsp;0.0143). The most significant reduction occurred in the 2-hour exposure group with CDP treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), highlighting CDP-cytidine's role in lowering adrenaline levels across different exposure durations.\u003c/p\u003e\u003cp\u003eIn addition, beta-endorphin levels in rat serum after e-cig exposure, with and without CDP-cytidine, show significant results, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD. Both the 2-hour and 1-hour e-cig exposure groups had markedly higher beta-endorphin levels compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and p\u0026thinsp;=\u0026thinsp;0.0001, respectively). CDP-cytidine treatment reduced levels in the 1-hour exposure group, but the difference was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.0893), nor was it significant in the e-cig quitting group (p\u0026thinsp;=\u0026thinsp;0.7377). The 2-hour e-cig group had significantly higher levels than the 1-hour group (p\u0026thinsp;=\u0026thinsp;0.0015), and CDP treatment further lowered beta-endorphin levels across all groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with the largest reduction seen in the 2-hour exposure group. However, no significant differences were found between the CDP-treated and quitting groups.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eE-cig exposure and CDP treatment were examined for the first time on withdrawal-induced anxiety, inflammation, and serum hormone levels. This study shows that e-cig exposure alters nicotine, cotinine, adrenaline, and beta-endorphin levels in rats, affecting hormonal balance and behavior. These data also suggest that CDP-choline (cytidine) may reduce the adverse effects of e-cigs.\u003c/p\u003e\u003cp\u003eChronic exposure to e-cig in this study resulted in significantly elevated serum levels of nicotine, cotinine, adrenaline, and beta-endorphin. Nicotine and its primary metabolite, cotinine, are well-known to interfere with the normal functioning of the endocrine system by triggering the release of catecholamines such as adrenaline. This elevation in catecholamines, particularly adrenaline, leads to heightened stress responses and is consistent with previous studies linking nicotine exposure to increased oxidative stress and neuroinflammation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBeta-endorphin, a hormone linked to the body\u0026rsquo;s pain and stress response systems, was also significantly increased following e-cig exposure, particularly in the 2-hour group. This finding aligns with reports that nicotine can stimulate the release of endogenous opioids like beta-endorphins, which contribute to nicotine's reinforcing effects and addictive properties [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The elevated beta-endorphin levels observed in this study suggest that e-cig exposure may enhance the rewarding and addictive potential of nicotine, creating a cycle that reinforces dependency and exacerbates hormonal dysregulation in exposed animals [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eE-cig-exposed rats' nicotine, cotinine, adrenaline, and beta-endorphin levels were significantly reduced by CDP-choline for 3 weeks, especially in the 1-hour exposure and quitting groups. CDP-choline reduces these effects by restoring cell membrane phospholipid metabolism, increasing neurotransmitter production (especially acetylcholine), and improving neural plasticity [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, CDP-choline treatment notably reduced anxiety-like behaviors, as evidenced by the LDB results. These behavioral improvements coincide with reductions in neuroinflammation and oxidative stress [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCDP-choline also had a pronounced effect on the reduction of adrenaline and beta-endorphin levels, particularly in the quitting\u0026thinsp;+\u0026thinsp;CDP group. This suggests that CDP-choline may aid in the normalization of stress hormone levels post-exposure, which is critical for recovery from nicotine addiction and withdrawal symptoms [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The lower levels of these hormones observed in the CDP-treated groups likely contributed to the reduction in anxiety behavior, as excessive levels of adrenaline and beta-endorphins are associated with stress and mood disorders [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe control and CDP-treated groups had similar beta-endorphin levels, demonstrating the compound's potential to restore hormonal balance without causing abnormal hormonal reactions. This reinforces research indicating that CDP-choline reduces external stresses and maintains endocrine system homeostasis [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Multifaceted processes explain CDP-choline's protective properties. It is commonly known that CDP-choline is a precursor of phosphatidylcholine, a significant cell membrane component. This helps maintain membrane fluidity, release neurotransmitters, and repair neurons [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese findings have significant implications for the development of therapeutic strategies aimed at mitigating the harmful effects of e-cigs. CDP-choline\u0026rsquo;s ability to alleviate both biochemical and behavioral changes induced by e-cig exposure underscores its potential as a therapeutic agent for nicotine addiction and its associated neurological and hormonal disturbances. Future studies should explore its long-term efficacy and potential use in clinical settings for treating e-cig addiction and neurotoxicity in humans.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study provides strong evidence that chronic e-cig exposure leads to significant hormonal disruptions and neurobehavioral changes, including increased anxiety, and stress hormone levels. CDP-choline treatment was effective in reversing these effects by reducing serum nicotine, cotinine, adrenaline, and beta-endorphin levels, as well as improving behavioral outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and ARRIVE compliance\u003c/strong\u003e\u003cbr\u003eAll animal experiments were approved by the Research and Ethics Committee of the Faculty of Pharmacy at Applied Science Private University, Amman, Jordan (Approval No.: 2023-PHA-26). All procedures were performed in accordance with institutional guidelines and national regulations for the care and use of laboratory animals. This study is reported in accordance with the ARRIVE guidelines 2.0 to ensure rigorous and transparent reporting of animal research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cbr\u003eNot applicable. The manuscript does not contain any individual data or identifiable personal information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cbr\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cbr\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003cbr\u003eM.B. and A.K. conceptualized and designed the study. D.R. and S.A. contributed to data collection and initial drafting. M.B. and W.A. performed data analysis and interpretation. A.K., S.A., and M.A.S. contributed to literature review and manuscript editing. M.B. supervised the project and finalized the manuscript. All authors read and approved the final version.\u003c/p\u003e\n\u003cp\u003eAcknowledgment\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Applied Science Private University, Jordan for supporting this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eUnger, M. and D.W. Unger, \u003cem\u003eE-cigarettes/electronic nicotine delivery systems: a word of caution on health and new product development.\u003c/em\u003e Journal of thoracic disease, 2018. \u003cstrong\u003e10\u003c/strong\u003e(Suppl 22): p. 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S123-S131.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"e-cig exposure, anxiety-like behavior, addiction, CDP-choline, hormonal levels, neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-7184123/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7184123/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eE-cig use is increasingly popular, yet its impact on mental health, particularly anxiety and hormonal regulation during withdrawal, is understudied. Cytidine 5'-diphosphocholine (CDP-choline), a neuroprotective compound, may help manage withdrawal symptoms. This study explores the effects of CDP-choline on withdrawal-induced anxiety and hormonal imbalances from chronic e-cig exposure, focusing on serum levels of nicotine, cotinine, adrenaline, and beta-endorphins. Male Wistar rats were divided into five groups: control, e-cig-exposed, e-cigarette-exposed with CDP-choline, e-cigarette quitting with CDP-choline, and CDP-choline-only. E-cig exposure involved one hour, twice daily, five days a week for six weeks, followed by reduced exposure for three weeks. CDP-choline was administered for three weeks starting at week six. Behavioral tests, including the light and dark box (LDB) test, were conducted at baseline, during withdrawal, and post-treatment. E-cig exposure significantly elevated serum nicotine, cotinine, adrenaline, and beta-endorphin levels, while increasing anxiety-like behaviors. CDP-choline treatment effectively reduced nicotine and cotinine levels, particularly in the e-cig exposure\u0026thinsp;+\u0026thinsp;CDP group (p\u0026thinsp;=\u0026thinsp;0.0027) and the quitting\u0026thinsp;+\u0026thinsp;CDP group (p\u0026thinsp;=\u0026thinsp;0.0416). Additionally, CDP-choline substantially lowered adrenaline and beta-endorphin levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), reducing stress responses linked to withdrawal. In conclusion, CDP-choline mitigates the harmful effects of e-cig exposure by reducing hormonal imbalances and improving behavioral outcomes. These findings highlight its potential as a therapeutic option for managing e-cig-induced withdrawal symptoms.\u003c/p\u003e","manuscriptTitle":"Effects of E-Cigarette Exposure with/out CDP-Choline Treatment on Withdrawal-Induced Anxiety, and Hormonal Levels: Using Rat Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-20 13:43:59","doi":"10.21203/rs.3.rs-7184123/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-26T09:21:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-24T22:13:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-24T19:03:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"198134493716195594373853201112355352350","date":"2025-08-18T03:56:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164410250574788624410420308657327500765","date":"2025-08-14T17:29:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"228400886493476977720361417590660969801","date":"2025-08-14T13:36:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-13T06:30:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-13T06:26:12+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-31T07:23:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-24T10:49:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-24T10:45:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d51d0668-2f48-4771-af3d-ed737833c2c1","owner":[],"postedDate":"August 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53309378,"name":"Health sciences/Diseases"},{"id":53309379,"name":"Health sciences/Medical research"},{"id":53309380,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2025-12-01T16:09:46+00:00","versionOfRecord":{"articleIdentity":"rs-7184123","link":"https://doi.org/10.1038/s41598-025-26799-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-28 15:57:44","publishedOnDateReadable":"November 28th, 2025"},"versionCreatedAt":"2025-08-20 13:43:59","video":"","vorDoi":"10.1038/s41598-025-26799-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-26799-z","workflowStages":[]},"version":"v1","identity":"rs-7184123","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7184123","identity":"rs-7184123","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}