A Study on Morphofunctional State of Djungarian Hamster (Phodopus Sungorus) Lungs and Main Organs of Detoxification under Long-Term Exposure to E-Cigarette Aerosol

Introduction. Electronic nicotine delivery systems (ENDS) first became widely available in Europe in 2006 and turned into the most popular alternative to conventional cigarettes [1–6], especially among young people and adolescents of the age of 14 and older [1][7–10]. The most hazardous for the environment are the multi-component electronic cigarette (EC) liquids, which contain more than 30 toxic chemicals, including carbonyl compounds (aldehydes, ketones), metals, propylene glycol, and glycerol [11–16]. Moreover, carbonyl compounds include extremely toxic substances such as acrolein (maximum permissible concentration (MPC) in air 0.03 mg/m³) and formaldehyde (MPC 0.05 mg/m³), which irritate the mucous membranes of internal organs, as a minimum [17]. Also, acrolein causes mutagenesis in bacteria and yeast, exhibits mutagenic properties in mammalian cell cultures, and reduces activity of some components of the immune defence [18].

Studies examining the effects of two essential components of ENDS—propylene glycol and glycerin—show that propylene glycol, when aerosolized into the alveoli, boosts the destruction of surfactant, which ensures lung compliance and prevents lung tissue adhesion. This effect leads to pulmonary collapse (atelectasis), while adjacent areas undergo compensatory overstretching, causing emphysema. Propylene glycol is also considered a stimulating factor in the formation of squamous cell metaplasia of the larynx. In rats subjected to inhaling glycerin vapour, metaplasia of epiglottal epithelium was observed [19]. It is also known that glycerin heated in an e-cigarette up to 500°C transforms into acrolein mentioned above.

Apart from the main components, the ENDS also use a variety of flavouring agents. The Generally Recognized as Safe (GRAS) list includes flavouring agents for ingestion, however, the number of studies on inhaling the aerosols containing such substances is few. For example, it has been proven that inhalation of diacetyl contained in e-liquid causes obliterating bronchiolitis, the so-called “popcorn lung” disease (the phenomenon was identified in the early 2000s due to the mass illnesses of workers of popcorn factories) [20]. At some factories, diacetyl was replaced by acetylpropionyl, but even then, researchers from the US National Institute for Occupational Safety and Health (NIOSH) had to note significant deviations in the spirometry readings of workers at such factories [21][22]. In ENDS, these substances are used to impart buttery or caramel flavour, although pulmonary toxicity of acetylpropionyl to mammals has been proven. For example, rats exposed to it developed fibrosis and necrosis of the respiratory tract tissue; in mice exposed to the inhalation test (methacholine challenge test) more severe bronchoconstriction was observed [23]. On the whole, despite more than 7000 ENDS flavours resulting from the range of flavouring agents available on the market [24], only three studies have been published on the effects of flavouring agents on the organism [15][25][26].

A considerable amount of information can be found in the literature on the effects of electronic cigarettes and vapes on the organism of mammals. For example, it has been proven that exposure to ENDS vapour altered pulmonary function in rats [27]; reduced the conductivity of ion channels in sheep bronchial epithelial cells, and also led to mucus hyperconcentration [28]. A significant increase in pro-inflammatory cytokines was recorded in the lungs of rats [29], as well as development of the lipoid pneumonia [30]. It was also found that steaming with nicotine-free liquid affected the vital organs, which manifested in interalveolar septa thickening in the lungs of rats, filling the bronchial tubes with mucus obstructing the lumen, and lymphoid infiltration in bronchial tubes. In the kidneys, a threefold expansion of the lumens in the Shumlyansky-Bowman capsules was recorded, along with the damage of the renal corpuscles and the epithelium of the proximal tubules [31]. It was noted that numbers of bronchitis, pneumonia, and lung cancer have increased in dogs in large cities [32]. Several studies revealed the hepatotoxicity of e-cigarette liquids in rats [33]. However, the information on the biological effects of long-term use of e-cigarettes and vapes is insufficiently presented in the scientific literature.

The aim of the present study is to investigate the morphofunctional changes in the lungs and main organs of detoxification of Djungarian hamsters (Phodopus sungorus) during long-term exposure to e-cigarette and vape aerosols.

Materials and Methods. The research was conducted in 2021 at the Institute of Systematics and Ecology of Animals, Siberian Branch of Russian Academy of Sciences and the Experimental and Applied Biology Research and Educational Centre of Novosibirsk State Pedagogical University. The work was performed with the colony of laboratory Djungarian hamsters (Ph. sungorus) kept under 12L/12D photoperiod at a temperature of +22 ºC in standard cages (26 × 36 × 20 cm). Experimental animals (n = 36) were randomly divided into experimental and control groups: the experimental group included 10 males and 10 females; the control group included 8 males and 8 females.

During 80 days of experiment, hamsters in the experimental group were exposed to vapour from heating X-3 Yoghurt Pear liquid (PRIDE VAPE, Russia) containing glycerin and propylene glycol in a 70/30 ratio, nicotine at a dose of 3 mg/ml, and “Yogurt” and “Pear” flavouring agents. The vapour exposure consisted of inflowing cigarette vapour by means of a pump and filling with it the entire chamber two times with a 5-minute interval. After 10 minutes, the animals were removed from the containers. The procedure was performed daily, twice a day, with a 2-hour interval. Hamsters from the control group were delivered to the experimental room after removing the air with aerosol particles from the room, and then placed in the challenge chambers without exposure to ENDS vapour.

The experimental setup included a container for animal exposure to vapour. For this purpose, the plastic food containers of the dimensions 10 x 17 x 4 cm, and volume of 0.7 l, with tight-fitting lids were used. To optimize the process, two containers connected with 4 mm hoses (Boutte, Italy) were used simultaneously. A pump (Intex, China) with a volume of V~1.594 l was used to pump aerosol into the chambers.

To study the morphofunctional state of the lungs and main organs of detoxification, the lungs, kidneys, and livers of the animals from both groups were collected after decapitation and fixed in 10% neutral formalin. Tissue samples of these organs were dehydrated and clarified in isopropyl alcohol in compliance with the tissue processing protocol. The test specimens were embedded in paraffin blocks, and afterwards the series of 6-μm-thick slices were cut using a Slee Medical CUT 5062 rotary semiautomated microtome (SLEE Medical GmbH, Germany). These slices were mounted on glass slides using a 1:1 protein-glycerol mixture. The slides were stained with Böhmer’s hematoxylin and eosin. Collagen distribution was determined using the Mallory method [34].

Histological preparations of the organs were examined under transmission light microscope Axio Imager M2 with AxioVision Z2 M2 image analysing module (Carl Zeiss, Germany). Images were taken using an AxioCam HR CCD camera with Zen Lite software (Carl Zeiss, Germany). All images contained a scale bar.

Statistical data were processed by calculating arithmetic means (x) and their errors (Sx). Differences in parameters between the experimental group compared to those in the control group were assessed using the Student’s t-test and were considered significant at p ≤ 0.05. All calculations were performed using commonly accepted formulas and Microsoft Excel 2010.

Results. The morphological pattern of the lung parenchyma of animals from the control group generally corresponded to the normal structure of this organ (Fig. 1 a, b, c). In the preparations stained with Böhmer’s hematoxylin and eosin, bronchi of various calibres were clearly visible (Fig. 1 a, c). In large bronchi, multilayered and columnar structure of epithelium was observed (Fig. 1 c). In the lumen, mucous secretion and weakly expressed desquamation of the epithelium were identified mainly at the site of transition of the terminal bronchioles into respiratory ones. Intralobular blood vessels were filled with blood plasma. The morphological pattern of the alveoli, which included capillary endothelial cells, type-1 and –2 alveolar cells, and macrophages, corresponded to the norm (Fig. 1 b, c) [35-36].

In the examined lung tissue specimens of animals from the experimental group, changes affecting the lung parenchyma and interstitium were detected. In the specimens, alternating foci of atelectasis and emphysema were visible (Fig. 2a). Complexes of desquamated epithelial cells were visible in the lumen of the bronchi and bronchioles (Fig. 2a, b). The epithelium of the mucous membrane had lost its multilayered structure. At high magnification, the columnar epithelial cells were noticed to lose their cilia. In the lamina propria of the mucous membrane, signs of edema and swelling of the fibrous components of connective tissue were visible. Blood vessels of the lamina propria were excessively dilated, filled with plasma and blood cells. Among the latter, leukocytes were identified (Fig. 2c).

The interalveolar septa and the walls of the alveolar sacs were thickened due to plasma impregnation and capillary congestion (Fig. 2a). Macrophages, segmented neutrophils, and lymphocytes were found in the alveolar lumens (Fig. 2c). A typical feature found in the lung specimens of animals from the experimental group was an excessive increase in the number of type-2 alveolar cells (Fig. 2c). Among type-1 epithelial cells, cells with pyknotic nuclei were often found. Capillaries and venules were congested. Perivascular connective tissue was loose and edematous. Endothelial cells of the alveolar capillaries were swollen. The nucleus was large, with a high content of heterochromatin, as a rule, and was bulged into the lumen of the capillaries (Fig. 2d).

A typical feature of lung tissue in animals from the experimental group was localization of homogeneous black substance among bronchial epithelial cells and in the interstitium of the alveolar sacs (Fig. 3a). A histochemical reaction for collagen according to Mallory method revealed intense staining of slices for this protein in the stroma at the periphery of segmental, intrasegmental, and intralobular bronchi, arteries, and veins of the lung. In the interstitium of the lung tissue, the reaction for collagen was moderate (Fig. 3b, c).

When studying kidney specimens of hamsters from the experimental group, a statistically significant decrease in all studied parameters of the renal glomeruli was found compared to the control group, which indicates a decrease in the functional activity of the kidneys in animals exposed to vapour (Table 1).

The glomerular vessels were excessively dilated, filled with plasma and blood cells. Endothelial and mesangial cells were swollen, erythrocytes had signs of sludge phenomenon (Fig. 4b, d). A typical pathomorphological feature of kidney specimens of animals from the experimental group was a high content of nephrothelial cells with signs of lethal damage in all parts of the nephron. As a rule, the cytoplasm in dying nephrocytes does not stain with hematoxylin and eosin, the nucleus is hyperchromatic, the nuclear membrane is not identifiable (Fig. 4a, с). In the lumen of the distal and proximal convoluted tubules, as well as in the cytoplasm of nephrocytes, a fine granular black substance was noticeable (Fig. 4c). This sign was especially pronounced in the lumen of the collecting ducts and among the epithelial cells of this part of the nephron (Fig. 4c).

At the light optical microscopy, hepatocytes of the centrilobular and periportal zones of the liver lobule of animals from the experimental group differed by tinctorial properties [37]. The cytoplasm of light hepatocytes was intensely basophilic and filled with oxyphilic material (Fig. 5a, c). In liver specimens of hamsters from the experimental group, stained with hematoxylin and eosin, compared to the control group, the predominance of hepatocytes with pycnotic nuclei in the organ parenchyma was clearly visible (Fig. 5a, b). Upon detailed examination of the specimens, it became obvious that such cells were hepatocytes dying by apoptosis. Their distinctive features were pycnosis of the cells and margination of chromatin in the nucleus (Fig. 5c). The predominance of such cells in the centrilobular zone, responsible for detoxification, suggested that the liver of animals in the experimental group experienced a functional load that was beyond the physiological optimum, and its adaptive resources were not capable of providing an adequate response to the effect of toxic substances (Fig. 5c).

Discussion and Conclusion. The results of the study provide every reason to consider that the chosen design of hamster exposure to the vape aerosol results in structural and functional changes in the lungs, liver, and kidneys. Damage to the bronchial epithelium, alveolar epithelium, and microcirculatory endothelium was detected in the lungs. Light microscopy data do not allow us to identify damage to the structures of the air-blood barrier; however, the gross morphological changes detected in this compartment indicate a disruption in the structural and functional organization of the basal membranes of capillaries and epithelial cells. This is known to lead to impaired microcirculation in the lungs, increased permeability of pulmonary capillaries, and the development of pulmonary edema. This may explain the presence of interstitial and alveolar edema and distelectasis (alveolar collapse alternating with expansion) in the lung parenchyma, which develop with the participation of leukocytes and macrophages [38–39].

In accordance with complementarity principle, which postulates the unity of structure and function, the detected hemodynamic changes lead to the disruption of ventilation-perfusion relationships in lung tissue and to gas exchange disorder. The high level of type-2 alveolar cells in the tissue can be explained by an increase in their proliferative activity in response to damage to the alveolar epithelium by substances contained in the vape aerosol. In addition to their role in surfactant synthesis, type-2 alveolar cells are deemed to be ensuring the detoxification. The combination of the detected lung changes suggests the pathological features very similar to acute respiratory distress syndrome.

Excessive dilation of glomerular capillaries in the absence of hypercellularity and mesangial edema may be due to the direct impact of biologically active molecules on the capillary walls, leading to capillary damage and disruption of blood circulation. The presence of an unidentified homogeneous substance in the lumen of the proximal and distal sections of the nephron tubules and collecting ducts, suggests that this substance is transported through the glomerular basal membrane and enters the renal glomerulus from the general bloodstream. A decrease in the morphometric parameters of the structural components of the renal corpuscle in the kidney specimens of animals from the experimental group, compared to the control group, provides every reason to suspect a disruption of the renal filtration-reabsorption mechanism and the development of renal failure.

Despite the absence of the obvious signs of liver damage, the abundance of evolutionarily adapted to detoxification dark hepatocytes dying by apoptosis in the centrilobular zone of the liver lobule, indicates the involvement of toxic substances in this process, which enter the liver with the blood.