Open Veterinary Journal, (2026), Vol. 16(4): 2043-2053

Research Article

10.5455/OVJ.2026.v16.i4.9

Alterations associated with Karelia cigarette exposure and the protective role of Libyan Sidr honey in male albino rats

Eda M. A. Alshailabi*, Nura I. Al-Zail and Fatimah A. Mohammed

Department of Zoology, Faculty of Sciences, Omar Al-Mukhtar University, Al Bayda, Libya

*Corresponding Author: Eda M. A. Alshailabi. Department of Zoology, Faculty of Sciences, Omar Al-Mukhtar University, Al Bayda, Libya. Email: eda.muftah [at] omu.edu.ly

Submitted: 12/12/2025 Revised: 12/03/2026 Accepted: 24/03/2026 Published: 30/04/2026

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ABSTRACT

Background: Cigarette smoke (CS) contains multiple toxic substances, including nicotine, tar, carbon monoxide, and reactive oxygen species, which can induce oxidative stress and physiological disturbances. Chronic exposure may result in stress-related behavioral and hormonal changes.

Aim: This study aimed to evaluate stress-related behavioral manifestations, body weight changes, and serum cortisol levels in male albino rats exposed to Karelia Red cigarettes (KRC) and to explore the modulatory potential of Libyan Sidr honey (LSH).

Methods: Twenty-eight adult male albino rats were randomly assigned to four groups (n=7 each): Control (no treatment), Honey (LSH, 100 mg/kg/day, oral), Smoke (KRC exposure: 5 cigarettes/session, 5 sessions/day, 6 days/week for 4 weeks), and Protective (LSH + KRC). Behavioral observations were conducted using continuous video recordings, body weight was measured, and serum cortisol was assessed using ELISA. To minimize potential variability associated with hormonal cycles, only male rats were used.

Results: Rats exposed to KRC exhibited stress-related behavioral manifestations, reduced locomotor activity, decreased body weight gain, and elevated serum cortisol levels compared with the control group (p < 0.05). Rats in the protective group showed partial attenuation of behavioral disturbances and lower cortisol levels than those in the smoke-exposed group, while only a slight improvement in body weight gain was observed. These findings suggest a modulatory effect of LSH against CS-induced alterations.

Conclusion: LSH mitigated some stress-related behavioral and physiological effects of KRC exposure. These findings support the potential of LSH as a natural agent for modulating smoke-induced stress responses and provide preliminary experimental insights with potential translational relevance. Behavioral observations were descriptive and exploratory and should not be interpreted as quantitative measures. Future studies should incorporate standardized behavioral scoring using a structured ethogram for rigorous assessment.

Keywords: Albino rats, Behavioral alterations, Cigarette smoke, Cortisol, Libyan Sidr honey.

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Introduction

Cigarette smoke (CS) contains over 4,000 toxic compounds, including nicotine (NCT), tar, carbon monoxide (CO), heavy metals, and reactive oxygen species, making it a serious public health risk (Ambrose and Barua, 2004; World Health Organization, 2011). Chronic exposure to CS has been associated with oxidative stress, inflammation, and systemic physiological disturbances. These factors collectively contribute to tissue injury and functional impairment across multiple organs (Rom et al., 2013; Diniz et al., 2013). Experimental and epidemiological studies have demonstrated that these toxic components can adversely affect the central nervous system, leading to alterations in behavior, cognition, and stress regulation (Peters et al., 2008; Wang et al., 2024). Among the constituents of CS, NCT plays a central role in modulating neuronal activity by interacting with nAChRs, thereby influencing neurotransmitter release and behavioral responses (Brody, 2006; Omotoso et al., 2017). CO reduces oxygen delivery to tissues by forming carboxyhemoglobin, which may exacerbate neural hypoxia and contribute to stress-related behavioral abnormalities (Schick and Glantz, 2005). These mechanisms highlight how CS exposure can disrupt neuroendocrine balance and promote behavioral alterations, particularly under chronic exposure conditions (Ufele, 2017). Behavioral disturbances induced by CS exposure have been documented in animal models, including increased locomotor activity, agitation, anxiety-like behavior, aggression, and altered social interactions (Simpson et al., 2016; Hammad et al., 2023). Such behavioral changes are often accompanied by activation of the hypothalamic–pituitary–adrenal axis, resulting in elevated circulating cortisol levels as a physiological stress marker (Vining et al., 1983; Raber et al., 2021). Together, behavioral observations and stress hormone measurements provide a useful framework for assessing the impact of CS on Neurobehavioral function.

Natural products with antioxidant and stress-modulating properties have attracted increasing attention as potential protective agents against smoke-induced toxicity (Vallianou et al., 2014). Honey, a natural substance rich in phenolic compounds, flavonoids, vitamins, and trace elements, has antioxidant, anti-inflammatory, and immunomodulatory activities (Ali et al., 2020; Al-Shahed et al., 2020). In particular, Sidr honey, derived from Ziziphus species, has been shown in experimental studies to mitigate oxidative stress and improve physiological outcomes in various toxicity models (Al-Himyari, 2009; Yahaya et al., 2020). However, data on the effects of Libyan Sidr honey (LSH) on behavioral alterations associated with CS exposure remain limited, warranting further studies to accurately evaluate its protective role.

Therefore, the present study was designed to evaluate behavioral alterations associated with Karelia Red cigarettes (KRC) exposure in male albino rats and to assess the potential protective role of LSH.

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Materials and Methods

Animals

Twenty-eight adult male albino rats (Rattus norvegicus) aged 10 weeks and weighing 180–200 g were obtained from the animal house of the Zoology Department, Faculty of Science, University of Omar Al-Mokhtar, El-Beyda, Libya. Rats were housed in standard cages at 22°C ± 2°C with a 12:12 hours light/dark cycle. Animals were allowed 3 weeks of acclimatization before the experiment, with ad libitum access to standard rat food and water. All procedures complied with the national and institutional guidelines for animal care. To minimize potential variability associated with hormonal cycles, only male rats were used.

Chemicals

LSH was purchased from Sayyid Abdulwahd village, Jabal al Akhdar, Libya, and evaluated at the Omar Al-Mokhtar University Center Laboratory. Freshly prepared honey (100 mg/kg body weight) diluted in an equivalent volume of distilled water (0.5 ml) was administered daily via oral gavage (Kolawole et al., 2015; Alshailabi et al., 2024). KRC were obtained from local supermarkets and used in the smoke exposure experiments.

Experimental design and grouping of animals

Rats were randomly assigned to four experimental groups (n=7 per group) as follows:

1. 1. Control group (Control): Rats were maintained under standard laboratory conditions without CS exposure or any treatment.
2. 2. Honey group (Honey): Rats received LSH orally at a dose of 100 mg/kg body weight per day by diluting in 0.5 ml of distilled water for 4 consecutive weeks, according to previously published animal studies (Kolawole et al., 2015).
3. 3. KRC group (smoke): Rats were exposed to KRC for only 4 weeks.
4. 4. Protective group (Protective): Rats received oral LSH for 2 weeks before KRC exposure and concurrent honey administration throughout the 4 weeks of smoke exposure.

CS exposure protocol

Rats in the smoke-exposed and protective groups were exposed to CS using a smoke exposure system that was locally designed. The system consisted of a glass exposure chamber (80 × 30 × 40 cm), whose dimensions were adopted from a previously published protocol (Ahmadnia et al., 2007). A bee smoker connected to the chamber via a tube was used to generate CS, forming a modified exposure setup specifically developed for this study (Alshailabi et al., 2023). The exposure chamber was cube-shaped, and a removable cover allowed for unforced exchange of fresh air to prevent hypoxia.

Five cigarettes were burned sequentially during each exposure session. Each CS exposure lasted for 5 minutes, followed by a 10-minute ventilation period, and this cycle was repeated five times per day. The protocol was conducted 6 days per week for 4 consecutive weeks, according to previously established procedures (Khalaf and Mostafa, 2012; Abdalally et al., 2021; Alshailabi et al., 2024). The same number of cigarettes, exposure duration, and session frequency were used in all sessions to maintain experimental consistency. The exposure chamber was cleaned and ventilated between sessions to prevent smoke accumulation and ensure uniform exposure conditions.

Although the CO, NCT, and particulate matter (PM) concentrations were not quantitatively measured, the smoke exposure was standardized and visually monitored to maintain a relatively uniform smoke density throughout the experiment. All smoke-exposed animals were exposed to identical conditions, ensuring reproducibility and comparability between groups.

Honey administration

LSH was freshly prepared daily by diluting in 0.5 ml of distilled water and administered orally via gastric gavage at a dose of 100 mg/kg body weight (Kolawole et al., 2015). In the protective group, honey administration started 30 minutes before each KRC exposure session and continued daily throughout the 4-week experimental period (Alshailabi et al., 2023). The selected dose was based exclusively on previously published animal studies demonstrating the biological and protective effects of honey, rather than on human consumption patterns (Kolawole et al., 2015; Alshailabi et al., 2023; Alshailabi et al., 2024).

Behavioral assessment

Behavioral manifestations were assessed using structured qualitative observation rather than standardized quantitative tests. This approach was chosen to document stress-related behaviors associated with CS exposure under the present experimental conditions. To reduce potential bias, continuous video recordings were obtained for all groups during and immediately after smoke exposure sessions and analyzed by a blinded observer. Behaviors were categorized using a structured ethogram adapted from Hamilton et al. (2009) including locomotor activity, exploratory behavior, grooming, freezing/immobility, posture, and social interaction. Observations were based on directly observable and reproducible phenomena without emotive or anthropomorphic descriptions. No formal behavioral scoring or statistical analysis of individual behaviors was performed. Therefore, these assessments are descriptive and exploratory, providing preliminary insights rather than quantitative measures of behavioral function.

Measurement of body weight

Body weight was recorded at the beginning (initial body weight) and end (final body weight) of the experimental period using a precision electronic balance (±0.01 g). The percentage change in body weight was calculated using the following formula, as previously described by Abd Allah et al. (2016):

Change in body weight (%)=[(Final weight − Initial weight) / Initial weight] × 100

Serum cortisol level

Blood samples were collected via the orbital sinus and allowed to clot for 10 minutes. Serum was stored at −80°C until analysis. Cortisol levels were measured using an established enzyme-linked immunosorbent assay method (Vining et al., 1983). Samples were collected between 9:00 AM and 10:00 AM to minimize circadian variation, and results are expressed as mean ± SEM.

Statistical analysis

Data were analyzed using one-way analysis of variance followed by Tukey’s post hoc test for multiple comparisons among the experimental groups. The normality and homogeneity of variance were verified before the analysis. Statistical analyses were performed using Minitab version 17, and significance was set at p < 0.05.

Ethical approval

Efforts were made to minimize stress and discomfort during CS exposure. Exposure durations were limited, ventilation periods were provided, and animals were continuously monitored to prevent hypoxia and ensure welfare, in accordance with institutional and international guidelines, and approved by the Al-Mukhtar Bioethics Committee and the Libyan Authority for Scientific Research (NBC: 007.A.24.14).

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Results

Behavioral observations

Behavioral manifestations across all experimental groups were qualitatively documented using continuous video monitoring. The observations focused on stress-related behavioral patterns rather than quantitative behavioral performance. Rats in the control and honey-treated groups exhibited normal locomotor activity, exploratory behavior, grooming, and social interaction throughout the experimental period, with no stress-related manifestations. In contrast, KRC-exposed rats demonstrated clear stress-associated behavioral manifestations during exposure sessions. These included reduced locomotor and exploratory activity, altered posture, increased vocalization, aggregation away from the smoke source, and transient freezing or immobility. Increased urination and defecation were also observed during repeated exposure sessions, consistent with acute stress responses. In the protective group, behavioral manifestations were initially similar to those observed in the smoke-only group; however, locomotor activity, grooming behavior, and social interaction patterns progressively approached those of the control group with continued honey administration. Stress-related behaviors were less pronounced in the smoke group than in the non-smoking group, suggesting a partial attenuation of smoke-associated behavioral manifestations (Table 1). All behavioral findings were derived from qualitative assessment of video recordings and should be interpreted as descriptive indicators of stress-related behavioral responses rather than definitive measures of behavioral dysfunction. Representative figures illustrate altered posture, aggregation behavior, and reduced exploratory activity in smoke-exposed rats compared with control and honey-treated groups (Figs. 1–8). These figures are provided for descriptive purposes only and do not represent the results of quantitative behavioral analysis.

Table 1. Summary of the behavioral observations in the experimental groups.

Fig. 1. Smoke-exposed rats’ behavior included abnormal posture, animals moving randomly, jostling, increased vocalization, and physical interaction with whole-body shakes (A & B).

Fig. 2. Smoke rat population gathered in one place away from the smoke.

Fig. 3. Smoked rats trying to flee and looking for a vented location when the cover was opened.

Fig. 4. After repeating the smoke process, the same behavior of smoke rats gathered in one place away from the smoke, with increased urination and defecation.

Fig. 5. After the first week, the animals tended to calm down and gather away from the source of smoke. (A, B, and C).

Fig. 6. After 1 week, some rats began to search for the smoke source (A and B).

Fig. 7. After the second week of CS exposure, the smoke rats (C) showed yellowing of their hair and gross changes in external appearance, as associated with other groups (A: control, B: honey, and D: protective groups).

Fig. 8. After the second week of CS exposure, the protective rats showed greater movement and increased observable locomotor activity associated with the CS group.

Changes in body weight

No significant difference in the initial body weight was observed among the experimental groups (p > 0.05), indicating comparable baseline conditions (Table 2). At the end of the experimental period, the control and honey-treated groups did not differ significantly in terms of body weight (p > 0.05). In contrast, rats in the smoke group showed a significant reduction in final body weight (158.57 ± 7.6 g) compared with the control group (243.9 ± 12.84 g, p < 0.05). Pretreatment and concurrent administration in the protective group partially mitigated smoke-induced absolute weight loss, with a final mean body weight of 177.00 ± 3.71 g, which was significantly higher than that of the smoke group (p < 0.05) but did not fully reach control levels.

Table 2. Initial and final body weights and percentage changes.

Smoke-exposed rats displayed a significant decline in weight gain (17.76% ± 0.77%) compared with controls (24.29% ± 1.28%, p < 0.05). The protective group showed a slightly lower percentage change (17.00% ± 0.37%) than the smoke group; however, this difference was not statistically significant, indicating that compared with smoke exposure alone, LSH partially prevented absolute weight loss but did not significantly affect relative weight gain.

Serum cortisol level

The smoke group exhibited a significant elevation in serum cortisol levels compared with the control group, indicating the activation of the stress response (p < 0.05). The mean cortisol concentration in the smoke group was 6.42 ± 0.23 ng/ml, compared with 2.74 ± 0.09 ng/ml in the control group. Rats treated with LSH alone (Honey group) exhibited cortisol levels comparable to controls (p > 0.05), with a mean concentration of 2.49 ± 0.26 ng/ml. Notably, the protective group, which received honey before and during smoke exposure, demonstrated a significant reduction in cortisol levels compared with the smoke group (4.35 ± 0.16 ng/ml, p < 0.05). Although cortisol concentrations in the protective group were partially normalized, they remained slightly elevated relative to controls (Table 3).

Table 3. Average cortisol levels in the experimental groups.

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Discussion

Exposure to KRC caused male albino rats in the smoke group to exhibit abnormal postures, erratic movements, jostling, vocalizations, fighting, and attempts to escape from smoke exposure. Although this study provides qualitative observations of stress-related behaviors recorded using predefined operational categories (locomotor activity, exploratory behavior, grooming behavior, freezing/immobility, posture, and social interaction) adapted from Hamilton et al. (2009) to obtain measurable data, future studies should include quantitative behavioral scoring using standardized paradigms such as the open field test or elevated plus maze. These observations are consistent with previous findings in animals exposed to SS, indicating increased anxiety-like behaviors and hyperactivity (Alaie et al., 2025; Kurawa, 2024). Moreover, NCT in CS stimulates nAChRs and triggers the release of neurotransmitters such as dopamine, norepinephrine, acetylcholine, and glutamate, which may be involved in the observed hyperactivity and cognitive-related effects, as suggested by previous studies (Singer et al., 2004; Benowitz, 2008; Moreland-Capuia, 2019). Chronic exposure to CS also disrupts neurotransmitter balance in key brain areas, such as the hippocampus and amygdala, which regulate learning, memory, and fear-related behavior; however, these neurobiological mechanisms were not directly assessed in the present study (Davidson, 2002; Bannerman et al., 2004; Alhusban et al., 2023; Hammad et al., 2023). Additionally, rats in the smoke group experienced hair discoloration and decreased body weight, likely due to the combined effects of NCT, tar, and other CS constituents (Al-Delaimy et al., 2002; Laborada and Cohen, 2021). Body weight reduction may also reflect decreased appetite, increased metabolism, and elevated energy expenditure mediated by sympathoadrenal activation (Chen et al., 2005; Audi et al., 2006; Abdul-Ghani et al., 2014). These findings are consistent with those of prior studies demonstrating that CS and NCT exposure can negatively affect energy balance and metabolic regulation (Gupta et al., 2006; Ojeka et al., 2024).

Serum cortisol levels were significantly higher in the smoke group than in the control group, reflecting the activation of the hypothalamic–pituitary–adrenal axis due to stress induced by CS exposure (Matta et al., 1997; Mendelson et al., 2008; Agaga et al., 2023). Elevated cortisol levels may further influence behavior, cognition, and metabolism, including glucose regulation, insulin sensitivity, and bone metabolism (Raff et al., 1999; Sánchez et al., 2000; Badrick et al., 2007). These observations underscore the importance of minimizing stress and discomfort in experimental animals, which was addressed in this study by limiting exposure durations, providing ventilation periods, and continuously monitoring animal welfare.

Importantly, rats receiving LSH before KRC exposure (protective group) exhibited improved behavioral activity, attenuated reductions in body weight, and moderated cortisol levels compared with those exposed to smoke. However, these parameters did not fully return to control values, indicating that honey had a partial rather than complete protective effect. Such protective effects may be attributed to the antioxidant properties of honey and its ability to reduce oxidative stress (Hasanin et al., 2017). In addition, honey has been reported to modulate brain-derived neurotrophic factor, which may contribute to learning, memory, synaptic plasticity, and neuroprotection, as suggested by previous studies; however, these pathways were not directly examined in the present study (Al-Himyari, 2009; Mustafa et al., 2019; Yahaya et al., 2020). Moreover, honey contains phenolic compounds that may enhance neuronal function and improve resilience against CS-induced oxidative damage (Adgaba et al., 2017).

Overall, this study demonstrates that LSH partially mitigates the behavioral, metabolic, and hormonal disturbances caused by CS in male albino rats. Future research should incorporate quantitative behavioral assessments using standardized ethograms or scoring systems. In addition, longer exposure periods, dose-response analyses, and detailed biochemical and neurophysiological measures are recommended to strengthen the translational relevance of these findings.

This study has some limitations. First, the absence of direct measurements of neurochemical markers, oxidative stress indices, and precise smoke exposure metrics (CO, PM, and NCT) was noted. Moreover, behavioral observations were descriptive and qualitative, rather than based on standardized quantitative tests, providing exploratory insights into stress-related behaviors. Future studies should incorporate these measurements and examine the preventive and therapeutic effects of Sidr honey to elucidate the underlying mechanisms.

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Conclusion

KRC exposure induced stress-related behavioral alterations, elevated serum cortisol levels, and reduced body weight in male albino rats, indicating significant physiological and hormonal disturbances. LSH administration partially mitigated these effects, demonstrating exploratory protective potential on behavioral, metabolic, and hormonal parameters. These findings suggest that LSH may possess antioxidant and stress-modulating properties capable of attenuating some CS exposure-related adverse outcomes. However, the behavioral observations were qualitative and descriptive, and certain experimental limitations—such as the absence of standardized behavioral scoring and precise neurochemical measurements—should be acknowledged. Therefore, while these results provide preliminary evidence for the potential benefits of LSH, further well-controlled experimental and clinical studies are necessary to confirm its efficacy, determine optimal dosage regimens, and elucidate underlying mechanisms.

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Acknowledgments

The authors would like to sincerely thank the staff of the Zoology Department of Omar Al Mukhtar University for their valuable support.

Conflict of interest

The authors declare that there is no conflict of interest.

Funding

This research received no specific grant.

Authors' contributions

Eda M. A. Alshailabi and Nura I. Al-Zail conceived and designed the study. Fatimah A. Mohammed performed the experiments. Eda M. A. Alshailabi, Nura I. Al-Zail, and Fatimah A. Mohammed contributed to data acquisition and analysis. Eda M. A. Alshailabi drafted the manuscript. Eda M. A. Alshailabi and Nura I. Al-Zail critically revised the manuscript. All authors have read and approved the final version of the manuscript.

Data availability

All data supporting this study’s findings are available within the manuscript. 

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