Research Article

Histopathological Studies of the Heart in Male Rats (Rattus norvegicus) Exposed to Tobacco and Electronic Cigarettes Smoke: Effect of Animals as Passive Smokers

Tyagita Hartady1,2*, Briliant Maharani2, Shafia Khairani1,2, Mas Rizky A. A. Syamsunarno1, Brian Christian Sarniem2

1Department of Biomedical Sciences, Faculty of Medicine, Padjadjaran University, Indonesia; 2Veterinary Medicine Study Program, Faculty of Medicine, Padjadjaran University, Indonesia.

Received | February 02, 2025; Accepted | March 15, 2025; Published | May 17, 2025

*Correspondence | Tyagita Hartady, Department of Biomedical Sciences, Faculty of Medicine, Padjadjaran University, Indonesia; Email: [email protected]

Citation | Hartady T, Maharani B, Khairani S, Syamsunarno MRAA, Sarniem BC (2025). Histopathological studies of the heart in male rats (Rattus norvegicus) exposed to tobacco and electronic cigarettes smoke: Effect of animals as passive smokers. Adv. Anim. Vet. Sci. 13(6): 1236-1243.

DOI | https://dx.doi.org/10.17582/journal.aavs/2025/13.6.1236.1243

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

It has been reported that almost 1.3 billion people worldwide use tobacco products (World Health Organization, 2024). Tobacco contains nicotine and is highly addictive, and tobacco use is a significant risk factor for cardiovascular and respiratory diseases, over 20 different types or subtypes of cancer such as lung, pharyngeal, oral, pancreatic, and many other debilitating health conditions (World Health Organization, 2024; Collatuzzo et al., 2024). Every year, more than 8 million people die from tobacco use (World Health Organization, 2024). Cigarettes sold on the market contain 1.1 mg to 1.8 mg of nicotine (Southeastern National Tuberculosis Center, 2012). The proposed limit of 0.70 mg of nicotine per gram of total tobacco is based on FDA analysis. Even though around 10 mg is contained in one cigarette, only 1–2 mg is absorbed into the body; the rest is wasted in the air (Aji et al., 2017). This causes the possibility of exposure to nicotine in passive smokers. Tobacco can also be deadly for non-smokers and kills second- hand smoker (World Health Organization, 2024). The high death rate due to conventional cigarettes containing nicotine has led to the continued use of electronic cigarettes. A research reported that electronic cigarettes are considered safer because they do not contain nicotine, affecting so many people switching to electronic cigarettes (Johar, 2016; Wackowski et al., 2016). There are many different types of e-cigarettes, the most common types of e- cigarettes are electronic nicotine delivery system (ENDS) and electronic non-nicotine delivery system (ENNDS). These systems heat a liquid to create aerosols that the user inhales. These so-called e-liquids may or may not contain nicotine (but do not contain tobacco). They also typically contain additives, flavors and chemicals that can be harmful to people’s health (World Health Organization, 2024). The substances in tobacco cigarettes and electronic cigarettes are not only dangerous for active smokers but also for passive smokers. Secondhand smoke is cigarette smoke exhaled by active smokers, and thirdhand smoke is the residue of cigarette smoke in the smoker’s surroundings, including furniture and even hair or animal fur (Food and Drug Administration, 2024). Pets such as dogs and cats spend most of their time near the floor, where cigarette smoke is concentrated. In addition, smoking activities are still found among poultry and livestock workers, which causes animals to be exposed to cigarette smoke. Therefore, animals can absorb harmful cigarette components and act as a reservoir for cigarette smoke residue on their body surfaces (Smith et al., 2017). Pathological abnormalities in tissue can be studied through histopathological analysis (Yang et al., 2021). This analysis assesses the inflammatory process or healing phase while observing the presence and distribution of degraded products from the surrounding tissue. Based on the belief that electronic cigarettes will have a milder health impact compared to tobacco cigarettes and the increasing prevalence, histopathological studies are needed. However, until now, organ histopathological studies on exposure to tobacco and electronic cigarette smoke in mice have still been carried out one by one and there is limited report about exposure to the transition from tobacco to electronic cigarettes. Because there have not been many negative impacts and research related to exposure to switching from tobacco to electronic cigarettes, this research needs to be carried out.

MATERIALS AND METHODS

Ethical Consideration

Ethics and animal welfare principles have been applied to rats according to the provisions of the Ethics Commission of the Faculty of Medicine, Padjadjaran University (No: 914/ UN.5.KEP/ EC/ 2024).

Experimental Design

Thirty-two male Rattus norvegicus rats aged ten weeks weighing 200g from Bandung Institute of Technology, were placed and subjected to acclimatization for six days with feed transition adjustments, namely Citrafeed® RatBio, laboratory temperature adjustments, routine cleaning of cages, and provision of water ad libitum and placement in spacious cages to ensure the rats were in a healthy condition and meeting animal welfare. After conditioning, the animals were randomly divided into four groups, each consisting of 8 rats, with groups namely group 1 acting as a control, rats without exposure to cigarette smoke. Group 2, the Tobacco Smoke Exposure / Conventional Cigarette Smoke (CCS) group, was exposed to tobacco cigarette smoke for three cigarettes/session with an estimated duration of exposure of 10 minutes and one day, carried out in 2 sessions (morning and evening); the exposure was carried out for 30 days. Group 3, the Electronic Cigarette Smoke (ECS) exposure group, was exposed to electronic cigarettes with liquid as much as 0.6 ml/session with an estimated duration of 10 minutes and one day, carried out in 2 sessions; the exposure was carried out for 30 days. Group 4 is a group with Transitional Cigarette Smoke Exposure (TCS) with exposure to tobacco smoke according to the CCS group for 15 days. They were then exposed to electronic cigarette smoke, according to the ECS group, for 15 days. Every session, there will be exposure to cigarette smoke with the vacuum machine cycle on for 5 seconds and rest for 30 seconds until the tobacco cigarette runs out. Smoke exposure was set at two pm with an air supply at 2 pm, creating a total rate of 4 ppm (Hage et al., 2017).

Histopathological Analysis

After necropsy, several tissue samples were taken from, lung, testicle, heart, liver, kidney and spleen, which were then fixed in 10% neutral buffered formalin with a composition of 100 ml formaldehyde (40%), 900 ml distilled water, 4 grams of sodium monobasic (Dey, 2018). Dihydrogen phosphate and 6.5 grams of disodium hydrogen phosphate for 24 hours. After that, dehydration was carried out using ethanol with a concentration of 70% for 2 hours. Next, rinse using toluene. The following stages, namely impregnation/infiltration and embedding with paraffin wax, were carried out simultaneously (Dey, 2018). After that, carry out the microtome procedure using a microtome knife microtome machine, slider, preparation holder, and knife slice thickness adjuster (2 μm). Staining was performed using Hematoxylin and eosin (H and E) to provide morphological imaging. After H and E was complete, cover glass was installed, and labeling is carried out (Dey, 2018). The histopathological observation of the samples was carried out using an Olympus CX 23 binocular microscope with 40x and 100x magnification and 10 fields of view to identify pathological changes due to exposure to tobacco smoke and electronic cigarettes. Digital documentation of the observation results was done using a ZEISS Axio motorized microscope equipped with a digital image driver directly connected to a computer to obtain accurate and detailed digital images. The process of calculating the extent of histopathological parameter damage using Image J software.

Histopathological Findings Parameters of Heart Organ

Gibson et al. (2021) stated that pathological conditions can be assessed using histopathological examination methods. In this study, histopathological examination was carried out using a scoring system consisting of five categories (Grade 0, 1, 2, 3, 4, 5) which refers to the histopathological findings of the heart organ, as explained by Boison et al. (2019) and Khairani et al. (2022). Histopathological change parameters assessed in this study include congestion, bleeding, tissue death (necrosis) in the left ventricle of the heart, fibrosis, and degeneration (Eliasyer et al., 2021; Andari et al., 2023; Akhgari et al., 2017). This assessment is based on the results of pathological observations attached to (Table 1).

 

Table 1: Histopathological assessment.

Grade	Percentage	Description
0	0%	There are no histopathological changes
1	<10% of cell tissue is affected and damaged	Few (<7) focal lesions
2	<20% of cell tissue is affected and damaged	There are several distinct lesions and increased mononuclear infiltration.
3	>20%-<75% of cell tissue is affected and damaged
4	>75%-<100% of cell tissue is affected and damaged	Multifocal to confluent lesions covering 75% of tissue and heavy leukocyte infiltration
5	=100% of cell tissue is affected and damaged

 

References: (Boison et al., 2019; Khairani et al., 2022).

 

Statistical Analysis

The results will be analyzed statistically using parametric statistics. A normality test was carried out using the Shapiro-Wilk test. The non-parametric Mann-Whitney U test is carried out for parameters with non-normal distribution results. Data will be visualized with plus or minus standard error with a minimum level of significance set at P> 0.05 (Oda, 2012).

 

Table 2: Semiquantitative parameters for histopathological scoring of the Heart Organ.

Group	Scoring of Histopathological Damage to the Heart Organ
	Myocardium					Total Lession
	Congestion	Hemorrhage	Degeneration	Necrosis	Fibrosis
CN	1 ± 0.18	0 ± 0	0 ± 0.16	0 ± 0	0 ± 0	1/25
CCS	1 ± 0a	1 ± 0.18a	1 ± 0.12a	1 ± 0.18a	0± 0.18	4/25
ECS	1 ± 0.16	0 ± 0.16	0 ± 0.18	0 ± 0.16	0 ± 0.16	1/25
TCS	1 ± 0a	1 ± 0.16a	1 ± 0.12a	1 ± 0.16a	1± 0.12a	5/25b

 

The data are presented as: CN (Control); CCS (Conventional Cigarette Smoke); ECS (Electronic Cigarette Smoke); TCS (Transitional Cigarette Smoke). Comparison (±) of histopathological damage scoring, compared with standard error (SE); a: The group with the most severe scoring in the comparison of one lesion; b: The group with the most severe total score in the comparison of all lesions. Congestion and hemorrhage are normal pathological findings in the heart. However, in exposure to tobacco and e-cigarette smoke, there were differences in the severity of congestion and hemorrhage lesions due to exposure between treatment groups.

 

RESULTS AND DISCUSSION

The results of this research treatment did not cause death in rats, and there was no high mortality rate due to exposure to tobacco and electronic cigarette smoke in animals. The treated rats showed clinical signs such as weaknes and inactivity and appeared depressed compared to the control rats. It produced microscopic findings that could be assessed from the degree of histopathological lesion damage. Histopathological lesion assessment using semi-quantitative parameters of the heart area, including the myocardium, is shown in (Table 2). The normality test results on five parameters obtained P> 0.000, which indicates that the data is not normally distributed. This is continued in non-parametric testing to compare the significance of damage between groups. In congestion lesions due to CCS, ECS, and TCS exposure, statistically significant differences were found in the CN group with CCS (P=0.025) and TCS (P=0.025). Hemorrhagic lesions due to CCS, ECS, and TCS exposure, there were statistically significant differences found in the CN group with CCS (P=0.009) and TCS (P=0.003). For degenerative lesions due to CCS, ECS, and TCS exposure, statistically significant differences were found in the CN group with CCS (P=0.015) and TCS (P=0.015). In addition, significance was found in the CCS group with ECS (P=0.046) and ECS with TCS (P=0.046). Necrosis lesions due to CCS, ECS, and TCS exposure had statistically significant differences in the CN group with CCS (P=0.009) and TCS (P=0.003). Fibrosis lesions due to CCS, ECS, and TCS exposure had statistically significant differences in the CN group with TCS (P=0.001). In addition, significance occurred in the CCS group with TCS (P=0.046) and ECS with TCS (P=0.015). There were differences in histopathological findings of lesions (congestion, hemorrhage, degeneration, necrosis, fibrosis) when comparing TCS exposure with CCS and ECS. The most severe damage comparison occurred in the TCS group, with a score of 5/30 of the total lesions. Histopathological finding lession shown in (Figures 1, 2, 3, 4 and 5).

 

 

Histopathological findings due to exposure to cigarette smoke and electronic cigarettes and their changes in this study were observed in the heart organ. Pathological findings and mechanisms of cardiovascular changes due to cigarette exposure are caused by oxidative injury, endothelial damage and dysfunction, increased thrombosis, chronic inflammation, hemodynamic stress, effects on blood lipids, insulin resistance, reduced oxygen flow by red blood cells, and arrhythmogenesis.

 

 

The constituents found in cigarettes that cause cardiovascular damage are oxidizing chemicals, carbon monoxide, volatile organic compounds, particulates, heavy metals, and nicotine (Benowitz et al., 2016). Pathogenesis of CCS exposure leading to cardiovascular injury is illustrated in Diagram 1.

 

 

The main cause of cardiovascular damage in e-cigarette users is endothelial cell dysfunction and angiogenesis. The main constituent of e-cigarettes that causes cardiovascular disorders is nicotine, which has the potential for crosstalk with macrophage activation (Esteban et al., 2022). According to Benowitz et al. (2017), pathological findings and mechanisms of cardiovascular changes due to exposure to e-cigarettes are due to the activation of nAChRs, which can cause damage, including increasing hemodynamic changes, endothelial dysfunction, insulin resistance, dyslipidemia, arrhythmogenesis, inflammation, and changes in the myocardium. In addition to nicotine, there are also constituents of e-cigarettes that cause cardiovascular disorders, namely propylene glycol and vegetable glycerin, which, when heated can form acetaldehyde, formaldehyde, propylene oxide, acetol, allyl alcohol, glyoxal, methylglyoxal, and acrolein which affect cardiovascular disease (CVD) (Benowitz, 2017). Pathogenesis of ECS exposure leading to cardiovascular injury is illustrated in Diagram 2.

 

Oxidative stress is more common in tobacco cigarette users than in e-cigarette users (MacDonald et al., 2019). The incidence of platelet activation, oxidative stress, and endothelial dysfunction in e-cigarette users will be lower than in conventional cigarette users (Oliveri et al., 2020), so in this study, organ damage due to exposure to e-cigarette smoke is the smallest compared to tobacco or conventional cigarettes. According to Wang et al. (2018), dual use of cigarette and electronic smoke exposure can have a greater potential for toxic substances from the use of tobacco and electronic cigarettes due to the addition of nicotine and combustion products from e-cigarettes. The use of e-cigarettes and tobacco cigarettes can cause blood clots in the heart, atrial fibrillation, arrhythmia, and heart enlargement. In addition, there is no significant reduction in the condition of carcinogens and toxic substances in dual-use compared to only using tobacco cigarettes (Wang et al., 2018). It can be proven that the level of damage that results from the use of both types of cigarettes (TCS) is greater than the use of CCS or ECS.

There is no significant reduction in toxic substances in double exposure (TCS) with tobacco cigarettes (CCS), which causes some lesions not to differ significantly in the TCS and CCS groups. The magnitude of damage in the CCS group is due to exposure to toxic substances in tobacco cigarettes and electronic cigarettes. Hazardous chemicals in cigarette smoke include TSNA, heavy metals, polycyclic aromatic hydrocarbons, and volatile organic compounds (VOCs) with carcinogenic properties. In addition, cigarette smoke also contains chemicals that contribute to the development of heart disease (e.g., carbon monoxide, arsenic, cyanide) and lungs (e.g., acrolein, acetaldehyde). Electronic cigarettes work by converting electronic cigarette fluid into an aerosol with a heating component containing toxic metals, one of which is cadmium. In addition, electronic cigarette fluid contains chemicals such as propylene glycol, glycerin, and flavorings, some of which are associated with negative health impacts if inhaled. Thus, the dual-use exposure pattern may be an important predictor of overall toxicant exposure (Coelman et al., 2022). The absence of regeneration supports the severity of TCS damage after two exposures to CCS and ECS in cardiomyocytes and the nature of cardiomyocytes, namely irreversible injury after damage or cell death (Xiang et al., 2024). Pathogenesis of TCS exposure leading to cardiovascular injury is illustrated in Diagram 3.

 

Histological examination of organs is the most important part of determining the molecular cause of damage to be able to explain pathogenesis. Histopathological findings due to exposure to conventional cigarette smoke, electronic cigarettes are not significantly different from exposure to transitional cigarette smoke (electronic and conventional). The results of this study provide an overview of the impact of exposure to tobacco and electronic cigarette smoke and its transition, so special attention is needed for animal owners to limit their animals’ exposure to cigarette smoke as passive smokers.

CONCLUSIONS AND RECOMMENDATIONS

The histopathological findings in this study further develop and clarify previous findings regarding the impact of animals as secondhand smokers, such as stroke, lung cancer, and heart disease. The histopathological findings of heart organ lesions (congestion, hemorrhage, degeneration, necrosis, fibrosis) in TCS exposure with CCS and ECS can further identify heart disease. The most severe damage was found in the TCS group because it received two exposures from tobacco cigarettes and electronic cigarettes. This is represented by the area of damage in TCS, which is wider than ECS and CCS

This study focuses on the heart due to its high metabolic activity and constant exposure to circulating toxins from inhaled smoke. The findings highlight the increased risk of cardiovascular disease in animals, particularly in environments with passive smokers. Animal welfare is also impacted, raising ethical concerns for pet ownership and farming. Stricter guidelines are needed to protect laboratory animals and pets from smoke exposure. Veterinarians should educate pet owners, and institutions should enforce smoke-free policies. The observed histopathological changes suggest this rat model is valuable for studying passive smoking’s effects on human cardiovascular health. Future research should explore interventions and the long-term consequences of smoke-induced cardiac pathology.

ACKNOWLEDGMENTS

The author would like to thank the Central Laboratory team, Department of Basic Medical Sciences, Veterinary Medicine Study Program, Faculty of Medicine, Padjadjaran University for facilitating the implementation of this research activity. Thank you to the Ministry of Education, Culture, Research and Technology of the Republic of Indonesia and 1491/UN6.3.1/PT.00/2024 Padjadjaran University for the funds and facilities provided by Dr. Drh.Tyagita,M.VSc, during the research.

NOVELTY STATEMENTS

This research evaluates the histopathological impact of exposure to tobacco cigarettes and electronic cigarettes, as well as the transition to animals as passive smokers. Other studies have not commonly examined the comparison of the impact of use and switching between the two types of cigarettes.

AUTHOR’S CONTRIBUTIONS

The author has done the research and writing of this article with the contribution of Collecting data, data analysis. Writing the manuscript Briliant Maharani and Brian Christian Sarniem; Collecting data and data analysis, Tyagita Hartady; Assembling research design and reviewing the manuscript, Tyagita Hartady; Data analysis and review manuscript, Mas Rizky A.A.Syamsunarno and Shafia Khairani.

Conflict of Interest

The authors declare no conflict of interests in this article.

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