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Review Article | DOI: https://doi.org/10.31579/2766-2314/147
*Corresponding Author: Basma Amer El-Sayed Alsaid, Department of Zoology, Faculty of Science, University of Zawia, Libya
Citation: Basma Amer El-Sayed Alsaid1*, Amina Ali Saleh Kalila1, Azab Elsayed Azab2. (2025), Review Article: Integrative Mechanisms of The Cerebellum., J, Biotechnology and Bioprocessing, 6(3): DOI: 10.31579/2766-2314/147
Copyright: © 2025 Basma Amer El-Sayed Alsaid, this is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Received: 08 April 2025 | Accepted: 21 April 2025 | Published: 02 May 2025
Keywords: Formalin, Body weight, Lung weight, Histopthological alterations of the lung, Mint aqueous extract, Amelioration, Adult male albino rats.
Background: Anatomy departments utilize formalin to harden museum specimens, and cadavers. Moreover, it is a component of pressed wood goods, paper, textile fibers, adhesives, plastics, carpeting, foam insulation, disinfectants, nail hardeners, and some finger paints. Additionally, it is used to stop germs from spoiling fish, fruits, milk, beverages, ice cream, sweetmeat, and spices. Because the respiratory epithelial cells are damaged and lose their ability to function, it leads in acute lung injury, a cytotoxic reaction in the respiratory system. The purpose of this study was to assess how formalin affected the adult male albino rats' body and lung weights as well as the histological structure of their lungs, and how this effect was mitigated by an aqueous extract of mint leaves. Materials and Methods: For this experiment, thirty adult male albino rats were procured from the Libyan Medical Research Centre located in the city of Zawia. Three equal groups were formed out of the animals. As a control group, Group I was given drinking water. Group II was the formalin group; for 30 days, they were given solely formalin at a dose of 10 mg/kg BW. Groups III were treated as a formalin + mint group, receiving 10 mg/kg BW of formalin for one hour, followed by 30 days of daily dosages of 600 mg/kg BW of mint extract. Gastric tubes were used to deliver all samples orally. On the 30th day, the animals were weighed, anesthetized, then scarified by cervical dislocation, and dissected. This was done 24 hours after the last dose. The lungs were extracted, weighed, and some of the lung tissue was preserved for 72 hours in 10% neutral buffered formalin, followed by dehydration and paraffin embedding. Subsequently, 5μm slices were made and stained with hematoxylin–eosin for histological evaluations. The weight of the body and lungs was reported as mean ± SE. One-way analysis of variance was used to conduct multiple comparisons (ANOVA). A value of p less than 0.05 was deemed significant. Results: The adult male albino rats' body weight significantly decreased (P<0.01) following the administration of formalin and formalin+mint, according to the data. On the other hand, rats given formalin+mint for 30 days showed a significant (P<0.01) increase in body weight as compared to the formalin group. Rats given formalin for 30 days showed an increase (P<0.01) in lung weight as a percentage of body weight when compared to the controls. On the other hand, compared to the formalin group, adult male albino rats given formalin+mint for 30 days experienced a significant (P<0.01) decrease in lung weight as a percentage of body weight. Control rats' lung sections underwent histological examination, which revealed normal pulmonary tissue architecture. When rats were given formalin, their lung tissues changed significantly in comparison to the control group. Co-administration of mint extract with formalin caused improvement in the lung tissues and restored the histoarchitecture to near normal as in the control group. Conclusion: It can be concluded that administration of formalin to rats significantly decreased body weight and increased lung weight as a percentage of body weight and a severe histopathological changed in lung tissues in comparison to the control group. While, treatment with mint improved these changed.
The lung is the essential organ of respiration and the organ that receives the entire cardiac output. Also, the lung plays an important role in host defense and regulation of circulating levels of biologically active materials by extensive surface of pulmonary vascular bed (Fishman et al., 1998, Mohamed et al., 2012).
Formalin is used in the plastics industry, as a fumigating agent in operating rooms, as a disinfectant for the preservation of surgical and pathological specimens, as well as for the coloring and hardening of celluloid, museum specimens, and cadavers in anatomy departments (Verma et al., 2016). It is utilized in products such as pressed wood items, paper, textile fibers, adhesives and plastics, carpeting, foam insulation, cosmetics, nail hardeners, disinfectants, and specific finger paints and cleaning solutions (Mamun et al., 2014). Some dishonest vendors have treated perishable goods with formalin to prevent spoilage. In order to preserve fish fresh, vegetables (like tomato and cucumber), fruits (like apple and grapes), milk, drinks, sweetmeat, ice cream, and spices from deteriorating due to germs, formalin is often and illegally employed (Restani et al., 1992).
In order to sell fish at auction or on the market, fishermen intentionally inject them with formalin (Kartikaningsih, 2008, Maramis et al., 2015). Formalin does not fulfill safety criteria when used as a preservation because it can react with chemicals in cells to affect their function, injuring cells, tissues, organs, and even the organism itself (Mahdi, 2008, Maramis et al., 2015).
Formalin can be obtained naturally or artificially and used as a food preservative in food and drink (Tomkins et al., 1989, Mamun et al., 2014). Exposure to formaldehyde induced a hepatotoxicity and haematotoxicity in human and experimental animals (Al-Sarraj and Al-Habity, 2013, Maramis et al., 2015, Verma et al., 2016, Elshaer and Mahmoud, 2017). Because formaldehyde is quickly absorbed from the gastrointestinal tract after ingestion and from the respiratory system after inhalation, it is dangerous to use as a preservative (Mamun et al., 2014). Verma et al. (2016) reported that individuals may encounter respiratory distress, rhinorrhea, and ocular discharge at direct exposure to formalin fumes.
A significant elevated risk of cancer, particularly nasopharyngeal carcinoma in humans, has been associated with formalin exposure at work (Hayes et al., 1986). In isolated instances, high formalin concentrations have also been connected to gastrointestinal cancer (Takahashi et al., 1986). Long-term formaldehyde exposure has been linked to a number of reproductive diseases, including aberrations that may result in infertility during sexual maturation, spermatogenesis, sperm viability, and count, as well as histologically harmful effects on testicular tissue (Razi et al., 2013).
Formaldehyde induces cytotoxicity in the respiratory tract, in the form of acute lung injury, which is caused by respiratory epithelial cell damage and loss of function (Mohamed et al., 2012). So, much attention is paid to the effects of formaldehyde on the respiratory system, especially, the histological alterations induced in the lung by exposure to formalin.
Herbal products enhance antioxidants. Natural antioxidant defenses are endogenous from reactive oxygen species and restore the ideal balance in the equation of reactive radicals (free radicals) (Al-Mamary et al., 2002). Therefore, recent studies are directed to the protective effects of plants and foods rich in antioxidants, such as green tea, mint, Garlic, and rosemary. Peppermint (Mentha piperita) is a perennial aromatic herb belonging to the Labiatae family and the Mentha genus. It has tremendous medicinal uses in different parts of the world (Baliga and Rao, 2010) because it contains. It contains more than 40 chemical compounds, including flavonoids such as oxetine, phenolic acids such as caffeic acid and rosmarinic acid, and its essential oil is rich in menthol (Anonymous, 1999, Baliga and Rao, 2010). Its antioxidants contribute to the prevention and treatment of diseases associated with oxidative stress because they remove free radicals and neutralize peroxide-stimulating ions (Sharma et al., 2007, Singh and Gupta, 2011). It is used peppermint relieves flatulence, nausea and vomiting, is an antispasmodic, carminative, antimicrobial, and treats intestinal inflammation and irritable bowel syndrome (Bouchra et al., 2003).
Over recent years it has been demonstrated that both peppermint and its constituents induce antioxidant, antispasmodic, anticarcinogenic, antitumorigenic, antiallergic, antiinflammatory, antimutagenic, anticancer, antinauseant, antiseptic, antilipid peroxidation, antiheadache and antiobesity properties (Shah and D’Mello, 2004, Mimica-Dukic N, and Bozin, 2008, Jain et al., 2011, Rita and Animesh, 2011, Yi and Wetzstein, 2011, de Cássia da Silveira et al., 2013).
2. Objectives:
The current study aimed to evaluate the effect of formalin on body and lung weights and histological structure in the lung of adult male albino rats and amelioration by mint leaves aqueous extract.
Formalin was purchased from Sigma chemical company.
Mint (Mentha piperita) was purchased from Al-Zawiya market, and mint juice was made by mixing 20 g of mint leaves with 200 ml of distilled water in a blender, then straining and filtering it to obtain the purified mint extract. Mint juice was given orally at a dose of 0.6 g/kg body weight for 30 days) Barbalho et al., 2011).
Forty-eight adult male albino Rats (180-200 g) obtained from Libyan Medical Research Centre in the city of Zawia were used for this experiment. The animals were maintained on 12 h light and dark cycle, at 25±2 ºC and 60%-70% humidity with standard pellets diet. Animal welfare and experimental procedures were strictly in accordance with the guide for the care and use of laboratory animals published by Clark et al., 1997.
The animals were equally divided into three groups Group I served as a control group and received drinking water. Group II served as a formalin group and received only formalin at (10 mg/kg) BW for 30 days. Groups III served as a formalin + mint group and received a formalin at 10 mg/kg BW, and after an hour were treated with the mint extract at doses of 600 mg/kg BW daily for 30 days. All samples were administered orally using gastric tubes.
After 24 hours of final dose on the 30th day, the animals were weighed, anesthetized, then scarified by cervical dislocation, and dissected.
The lungs were removed, weighed, and a portions lungs were fixed in 10% neutral buffered formalin for 72 h, dehydrated, and embedded in paraffin. Later, 5μm sections were prepared followed by staining with hematoxylin–eosin for histological assessments.
Body weight and lungs weight were expressed as mean ± SE. Multiple comparisons were performed by one-way analysis of variance (ANOVA). Value of p < 0>
Body weight was markedly decreased in all groups under investigation after administration of formalin and formalin+ mint in adult male albino rats. The data recorded in table (1) and figure (1) indicate a marked decreased (P<0>) in body weight in rats received formalin and formalin+ mint for 30 days compared with the controls. While, administration of formalin+ mint to adult male albino rats for 30 days caused a significant increase (P<0>) in body weight compared with the formalin group.
Lung weight as a percent of body weight was increased (P<0>) in rats received formalin for 30 days compared with the controls. While, administration of formalin+ mint to adult male albino rats for 30 days caused a significant decrease (P<0>) in lung weight as a percent of body weight compared with the formalin group (Table .1 & Figure .2)
Table. 1: Effect of administration of adult male albino rats to formalin and formalin+ mint on the body weight and lung weight (gm/100gm of body weight).
Groups Parameters | Control Mean ± SE | Formalin Mean ± SE | Formalin + mint Mean ± SE |
| Body weight (g) | 298.5 ± 4.60 | 34.8 ± 16.07**2 | 270 ± 2.63**## |
| Lung weight (g/100g B.W) | 0.715 ± 0.069 | 0.951 ± 0.015** | 0.787 ± 0.029## |
*: significant at (P<0>) compared to control group; **: significant at (P<0>) compared to control group;
#: significant at (P<0>) compared to formalin group; ##: significant at (P<0>) compared to formalin group.
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| Figure. 1: Effect of administration of formalin and formalin+ mint to adult male albino rats on the body weight. | Figure. 2: Effect of administration formalin and formalin+ mint to adult male albino rats on the lung weight (gm/100gm of body weight). |
Lung sections from the control rats revealed a normal pulmonary tissue architecture with clearly visible patent bronchial passageways and alveolar cavities, which included the alveolar sacs, alveolar ducts, and alveoli. The alveoli were polyhedral chambers with narrow walls that were encircled by a single layer of squamous epithelium. There was a thin layer of connective tissue in between each alveolar. There are thin and thick sections of the alveolar septum. Both type I and type II alveolar cells made up the alveolar septum. Type I alveolar cells were thin, squamous, and flat, and they covered the majority of the alveolar lining surface in the lung tissue of the control samples. Type II cells, on the other hand, were cuboidal secretary cells that were present at the angular junctions of the alveolar walls, scattered among the type I cells but with a tendency to converge at the septal junction. In order to clear inhaled particulate debris, alveolar macrophages that were present in the alveolar wall also moved into the lumen. The distribution of pulmonary vessels throughout the pulmonary parenchyma was normal (Figure 3).
Figure. 3: Histological structure of the lung of control adult male albino rats. The alveoli (White arrows) were thin-walled polyhedral chambers surrounded by single layered squamous epithelium (Green arrows), and alveolar sacs (AS). (A: X100, B: X400; C: X1000, H&E).
4.2. 2. Histological examination the lung of formalin administrated rats
When rats were 2. given formalin, their lung tissues changed significantly in comparison to the control group. Lung sections revealed abnormalities in the pulmonary cytoarchitecture, such as the alveolar walls being thickened in certain areas and obliterated in others. The alveolar septal veins also shown dilatation and congestion. Purulent exudates in the bronchioles, pulmonary fibrosis with thickened alveolar walls, enlarged alveolar walls, type II pneumocyte hyperplasia, cellular pyknosis, bronchiolar epithelia degeneration, a few collapsed alveoli, alveolar hemorrhage/edema, inflammatory cell infiltration, and foamy macrophage accumulation. Pneumocyte proliferation causes the alveolar wall to become more cellular, thickening the alveolar septa. Emphysema is demonstrated by the production of air space bullae and dilated interalveolar septal capillaries. Animals' lungs displayed vasculitis along with pulmonary blood vessel dilating (Figure 4).
Figure. 4: Histological structure of the lung of formalin administrated adult male albino rats. The alveoli (White arrows) The alveolar hemorrhage (Blue arrows), thickening the alveolar septa (Red arrows), inflammatory cell infiltration (Yellow arrows), Purulent exudates in the bronchioles (Yellow stars), Emphysema (Red stars), few collapsed alveoli (Green arrows), and alveolar sacs (AS). (E: X100, A, B, C, F, G& H: X400; D: X1000, H&E).
When formalin+mint was administered to adult male albino rats, histological analysis of the lung sections revealed that the control group's pulmonary tissue architecture was almost normal. The alveoli were polyhedral chambers with narrow walls that were encircled by a single layer of squamous epithelium. There was a thin layer of connective tissue in between each alveolar. There are thin and thick sections of the alveolar septum. Both type I and type II alveolar cells made up the alveolar septum. Type I alveolar cells in lung tissue were thin, squamous, and flat, and they covered the majority of the alveolar lining's surface. Type II alveolar cells, on the other hand, were cuboidal secretary cells found at the alveolar walls' angle junctions, scattered among type I cells but with a tendency to converge at the septal junction. In order to eliminate inhaled particulate debris, alveolar macrophages that were located in the alveolar wall also moved into the lumen. Alveolar minor infiltration of cells. There was evidence of dilatation and congestion in several alveolar septal veins (Figure 5).
Figure. 5: Histological structure of the lung of formalin+mint administrated adult male albino rats.The alveoli (White arrows) were thin-walled polyhedral chambers surrounded by single layered squamous epithelium (Green arrows), alveolar sacs (AS), and alveolar minimal cellular infiltrate. Dilatation and congestion were found in some of the alveolar septal vessels (A: X100, B: X400, H&E).
The current study was carried out to evaluate the effect of formalin on body weight and lung weight (g/100g of body weight) and histological structure in the lung of adult male albino rats and amelioration by mint aqueous extract.
Researches on humans and laboratory animals has demonstrated that both acute and long-term exposure to formaldehyde can cause cancer and respiratory damage (Lu et al., 2008, Turkoglu et al., 2008, ATSDR, 2010, Zhang et al., 2013).
The results of the current study showed a significant decrease in body weight of rats that received formalin compared to the controls. These findings run parallel with study of Tobe et al., 1989 who found that shown a reduction in the body weight of subjected to formalin. It is possible that this reduction in body weight was caused by insufficient consumption of food and water. Furthermore, Park et al. (2020) observed that mice exposed to 5.36 mg/m3 formaldehyde saw a significant reduction in body weight gain between day 4 and day 15. Rats and mice studies have demonstrated that formaldehyde exposure at concentrations between 3 to 400 ppm reduces food and water intake and body weight, while concentrations above 6 ppm in human and animal models alter pulmonary function and reduce body weight (Tesfaye et al., 2021). Egwurugwu et al. (2018) observed that the rats exposed to formalin displayed several physical symptoms, including altered eating patterns and decreased body weight. Compared to control F344 rats, the mean body weights of the F344 rats treated with formaldehyde were decreased (Ohtsuka et al., 1997). When pregnant mice were given formaldehyde, their body weight decreased in comparison to the control group (Merzoug and Toumi, 2017). According to a study (Aydin et al., 2015), rats exposed to formaldehyde at a level of 5.27 ppm experienced a drop in body weight relative to the control group; this drop in weight may have been caused by a decrease in hunger (Aydin et al., 2015, Merzoug and Toumi, 2017).
Repeated exposure to toxic substances leads to chemical stress that activates the hypothalamic pituitary adrenal axis, which leads to increased secretion of cortisol from the adrenal glands (Friedman & Lawrence, 2002), and the response to stress in humans has led to a 30
It can be concluded that administration of formalin to rats significantly decreased body weight and increased lung weight as a percentage of body weight and a severe histopathological changed in lung tissues in comparison to the control group. While, treatment with mint improved these changed.
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