Occupational and household formaldehyde is a common hydrophilic compound that is immediately retained through the lungs and, to a much lower extent, the skin. Health effects related to its exposure are pronounced when the body at sites like the eye, nose, skin, and throat has direct contact with the compound [1
]. Researchers have deduced the relationship between the health effects and range of exposures, with some individuals becoming symptomatic at low levels of exposure. A few people may have gentle uneasiness while others have moderate or no inconvenience at comparative exposures. Mean level exposures are at their most elevated in the clinical dissection room or morgue [3
]. Formaldehyde (FA) is a colorless, combustible and extremely reactive chemical at standard pressure and temperature [4
]. It is broken down in the air and highly stable in liquid [5
]. It rapidly diffuses in any tissues, e.g., the liver, through the oral or intraperitoneal route since it collaborates with various cell components [6
]. Formalin was first used as a fixative and treating liquid; however, these days it is used in every field of daily life. The most appalling use of formalin is as food additive [7
] and that’s why there is a drastic increase in human exposure to formalin intoxication. After intake, FA is readily absorbed from the gastrointestinal tract. In the liver, FA is largely metabolized to methanol and formate by aldehyde dehydrogenase 1 or mitochondrial aldehyde dehydrogenase 2, respectively. However in high concentrations of FA, toxicity arises in the hepatocytes [8
Research conducted on FA exposure to animals shows hepatotoxicity and abnormal histological alterations in the gastrointestinal tract [9
]. A low dose of FA has been shown to be mutagenic and carcinogenic and can manifest in a wide range of toxicities in different organs [10
]. Gastrointestinal cancer can also be caused by drinking water containing a high concentration of FA [11
]. In growing countries like India and Nigeria, the haphazard use of FA in lots of food items and drinking water has exposed a large percentage of citizens to a huge health hazards such as liver damage [12
]. The liver is a large, composite organ that performs very important tasks in sugar, fat and protein digestion. It functions in the detoxification of metabolic wastes like ammonia. Together with the spleen, it is associated with the obliteration of the remnants of the erythrocyte and the re-use of its constituents. Bile synthesis and secretion are also present in the liver, lipoproteins and plasma proteins synthesis, as well as coagulating factors. It maintains a steady level of blood glucose via glycogenesis, glycogenolysis and gluconeogenesis. The liver also plays a significant role in the elimination and detoxification of drugs. Therefore, xenobiotics (for example, liquor and numerous drugs), malnutrition, infection, and anemia, can induce liver damage [13
]. Hepatic damage is a common disease that mostly occurs as a result of oxidative stress and involves progressive growth from steatosis to hepatocellular carcinoma [14
Over 2 millennia, most Chinese medicines have made use of fungi for the management of a range of diseases [15
]. Traditional oriental therapies have also benefited greatly from medicinal mushrooms, and fungal metabolites are widely used in the treatment of diseases. [16
]. Additionally, mushrooms should not only be considered as food, as research has shown that they contain a lot of biologically active compounds [17
]. Mushrooms have numerous compounds with some biological significance. The extensive list incorporates polysaccharides, phenolics, proteins, polysaccharide–protein complexes, lipid components, and terpenoids, alkaloids, little peptides and amino acids, nucleotides and nucleosides [18
]. This extensive list refers to an extraordinary combination of organic properties, including cancer prevention agents [19
], antitumor [20
], antimicrobial [17
], immunomodulatory [21
], anti-inflammatory [22
], antiatherogenic [23
] and hypoglycemic activities [24
]. Gandoerma lucidum
(Lingzhi, Reishi), which has been used for quite a long time in Asian nations to improve wellbeing and advance life span, is widely perceived as a means of avoiding and treating many diseases, including malignant growth [25
]. As far back as 1986, the lethal dose (LD50
) has been reported to be 5000 mg/kg [26
]. In 2006, reports investigated the beneficial roles of G. lucidum
. Although the vast majority of persuasive information depends on laboratory and preclinical investigations, G. lucidum
has gained consideration in non-Asian nations [27
Investigations with refined G. lucidum triterpenes have indicated in vivo results, which can be used for drug development. However, the full use of G. lucidum preparation in corresponding and alternative medications is progressively beneficial because the specific component of G. lucidum could have synergistic or added substance impacts and could influence molecular signaling pathways and targets, finally prompting the destruction of malignant cells. This study is therefore performed to examine the protective potential of G. lucidum in formaldehyde-induced liver damage in experimental rats.
2. Materials and Methods
2.1. Fungi Material and Extraction
The whole basidiomata of G. lucidum was obtained from a village in Edo state. Identification was conducted at the Department of Agricultural Sciences, Joseph Ayo Babalola University. G. lucidum was air dried away from the direct sun rays and samples were milled to a total of 413 g of boorish powder. In total, 200 g of the quantity of coarse powder was soaked in 1 L ethanol for 72 h, decanted and concentrated, thus yielding a dark brown extract. The extract was weighed and stored in the refrigerator.
Thiobarbituric acid, 1-chloro-2,4-dinitrobenzene (CDNB), 5′,5′-dithiobis-2-nitrobenzoic acid (DTNB), xynelol orange, reduced glutathione (GSH), epinephrine and hydrogen peroxide (H2O2) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other chemicals and reagents were obtained from Cloud-Clone Inc., Wuhan, China.
2.3. Animals Care
Forty sexually matured Wistar strain male rats with the weight range of 150–200 g, were acquired in the animal colony, University of Ibadan, Nigeria. They were housed in a polycarbonate cage in an Assessment and Accreditation of Laboratory Animal care-certified animal facility and adherence to the protocol of the National Institute of Health on the Guide for the Care and Use of Laboratory Animals. Prior to acclimatization for 2 weeks, rats were kept under 12:12-h light:dark cycle and provided with NIH-07 diet and water ad libitum.
2.4. Experimental Design
Forty sexually matured Wistar strain male rats were divided into four groups of ten rats each and treated for thirty days (2 weeks of acclimatization inclusive) as described thus:
Group 1: were orally treated with 1 mg/mL distilled water.
Group 2: were exposed to 40% FA vapor environment for 30 min daily (the exposure was done by soaking 50 mL of FA in cotton wool and placed in a corner within the animal cage, thus exposing the animal to the vapor for a period of 2 weeks (40% FA at room temperature) [28
Group 3: were orally treated with 100 mg/kg ethanol extract of G. lucidum.
Group 4: were co-administered FA and 100 mg/kg ethanol extract of G. lucidum (1/50 of LD50). The route of administration of G. lucidum was oral and that of FA was the same as in Group 2.
Rats were then sacrificed 24 h after the last administration via cervical dislocation. Liver samples were excised, weighed, homogenized, and then processed for further experiments.
2.5. Determination of Liver Function Parameters
Blood samples were collected after sacrifice and plasma samples were obtained using the standard method. Liver function biomarkers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), triglycerides, total bilirubin, direct bilirubin, albumin and cholesterol were assayed according to the manufacturer’s procedure (Randox Laboratories, Crumlin, UK).
2.6. Estimation of Antioxidant and Oxidative Stress Markers
The excised liver samples were homogenized accordingly using a 50 mM Tris–KCl buffer at pH 7.4 consisting of 1.15% KCl, then further centrifuged at 12,000× g
for 15 min at 4 °C and afterward used for biochemical assays. Estimation of the protein concentration was done according to Bradford [29
]. Claiborne [30
] and Misra and Fridovich [31
] methods were used to determine the activities of Catalase (CAT) and superoxide dismutase (SOD). Glutathione-S-transferase (GST) activity and the GSH level were determined according to Habig et al. [32
] and Rotruck et al. [33
]. Meanwhile, the level of lipid peroxidation (LPO) was determined according to Jollow et al. [34
generation was determined according to the standard method of Wolff [35
]. All biochemical experiments were analyzed using a SpectraMax plate reader (Molecular Device, San Jose, CA, USA).
2.7. Assessment of Inflammatory Biomarkers
Myeloperoxidase (MPO) activity was determined according to the method described by Granell et al. [36
], whereas the nitrite level concentration was assessed using an established protocol [37
2.8. Determination of Proinflammatory Cytokines
Tumor Necrosis Factor (TNF-α), IL-1β and IL-6 concentrations in liver homogenates samples were assayed using rat TNF-α, IL-1β and IL-6 Elisa kits, respectively (Cloud-Clone Inc., Wuhan, China). A microplate antibody-coated plate was provided with the kit. All reagents, samples and working standards were prepared using standard procedures as provided by the kit manufacturers.
2.9. Histological Examination
Liver samples of rats that were removed were fixed with Bouin’s solution which was subsequently dehydrated in graded concentrations of alcohol. This was further cleared three times using xylene solution and was later embedded in paraffin wax. Microtome was then used to cut 4–5 mm of the paraffin waxed tissue on a slide and it was stained with haematoxylin (H) and eosin (E). The slides were then further viewed using a light microscope (Olympus CH; Olympus, Tokyo, Japan) and were snapped by pathologists.
2.10. Ethical Approval
All procedures involving animals performed in the study were performed in accordance with the ethical standards of our institution.
2.11. Statistical Analyses
Data were evaluated as mean ± SEM. Levels of statistical significance were analyzed with a one-way analysis of variance (ANOVA) which was further subjected to Bonferroni’s post hoc test using GraphPad Prism 6 software. p < 0.05 was considered significant.
The use of natural products in the prevention and management of various illnesses has prominently increased in the last few years [38
]. The present study established the promising chemopreventive potential of G. lucidum
in the liver, preventing liver damage caused by FA exposure. The reestablishment of unhealthy liver functions was evident by the remarkable loss of body weight, and a significant reduction in the liver organ weight which can result from shrinkage in the liver as seen in the FA-administered group (Table 1
), but this was prevented in the rats treated with G. lucidum
. The hepatoprotective potential of G. lucidum
against FA was investigated by determining ALT, AST and ALP. ALT is the important liver damage enzyme that catalyzes transamination reactions. The occurrence of conditions that can cause liver damage such as cancer, injury and hepatitis, will result in higher levels of this enzyme [14
]. AST and ALP, the biomarkers of liver damage, are cytosolic and mitochondrial enzymes whose levels are usually increased in cases of chronic illness and necrosis due to loss of hepatocellular integrity. These enzymes are involved in the transfer of α-amino groups from alanine and aspartate to the α-keto group of ketoglutarate to form pyruvate and oxaloacetate, respectively [39
]. As shown in figures, there is a significant increase (p
< 0.05) in the levels of these enzymes in the group administered FA when compared to the control. However, treatment with 100 mg/kg G. lucidum
significantly reduced the elevated levels, showing that G. lucidum
exhibits a protective role against FA-induced liver damage in rats. This study is related to that of Lakshmi et al. [40
] that showed the effects of Ganoderma lucidium
on hepatic damage induced by benzo(a) pyrene. The elevated liver function enzymes were significantly reduced by Gandoerma lucidum
Albumin is a measure of the synthetic function of the liver. A significant decrease in the albumin level in the FA-administered group could be traced to the reduction in protein synthesis that is an effect of FA. The carbonyl atom of FA reacts with the amino groups (nucleophilic sites) on the cell membranes forming hydroxymethyl amino acid derivatives [41
]. However, treatment with 100 mg/kg G. Lucidum
significantly increases the level of albumin. Cholesterol oxidation causes enzymatic increases in bile acids and contributes to hepatic cholesterol accumulation and hepatocellular injury. This is further explained by its significant increase in the FA-administered group as compared to the control. However, G. Lucidum
treatment reduced the elevated cholesterol levels significantly when compared to the control. Total direct bilirubin is also an indicator of the destruction of erythrocytes and the proper functioning of the liver, gallbladder and bile ducts, and is a potential marker for liver damage. Triglycerides were also increased in the FA-administered group as compared to the control. However, G. Lucidum
reduced the elevated cholesterol levels significantly when compared to the control. The liver index, the indicator of hepatic manifestation of metabolic disorders, was upregulated in the group administered FA, thus showing an impairment of the liver. However, upon administration of 100 mg/kg G. lucidum
to the group induced with 40% FA, there was a significant downregulation in the increased liver index.
When the body metabolism is impaired, an increase in the production of toxic molecules such as free radicals and antioxidants, known as free radical scavengers, are needed to reduce or neutralize the free radical formation [42
]. Our results show that FA has a direct effect on the hepatocytes and also an indirect effect through the circulatory and immune systems [43
]. The hepatic destruction caused by FA causes oxidative stress and produces reactive oxygen species (ROS), as shown in the significant increase in H2
and LPO, which are known to be oxidative stress markers, and also a decrease in GSH, GST, catalase and SOD, which are antioxidant markers. These observations are accordance to Payani et al. [44
] who reported that FA exposure significantly reduced the levels of enzymatic and non-enzymatic antioxidants. However, G. lucidum
significantly increases the activities and levels of these antioxidant markers. These results indicated that animals treated with G. Lucidum
cause a significant increase in the levels of antioxidant enzymes. These results are in line with the reports of other researchers that allude to the fact that Gandoderma lucidium
has antioxidant activities both in vivo and in vitro [45
]. These results indicated the hepatoprotective efficacy of G. Lucidum.
Myeloperoxidase is one of the most important molecules released after the recruitment and activation of phagocytes and it is involved in the production of oxidative stress. Additionally, proinflammatory cytokines activate iNOS during liver injury to abnormally producing NO that contributes immensely to the pathogenesis and evolution of liver damage. The present study shows a distinct increase in the activity and level of MPO and NO in FA-administered rats’ livers as compared to the control. However, the reduced level of MPO and NO following G. lucidum
treatment shows its potential to prevent inflammation in the liver of rats [48
TNF, IL-1β and IL-6 play a major role in the pathogenesis of liver damage. TNFs are majorly a group of proinflammatory cytokines known to perform a crucial role in the instigation of liver damage with evidence that oxidative stress might act in conjunction with endotoxins to augment TNF production [50
]. Interleukin 1β and 6 are potential biomarkers of acute or chronic liver toxicity. TNF, IL-1β and IL-6 are proinflammatory cytokines that are released into the bloodstream from the liver during hepatic toxic injury. Thus, biological agents suppressing these cytokines are known to have demonstrated huge therapeutic potential. As shown in our results, there was a significant upregulation in the levels of these cytokines in rat livers when administered FA. The significant downregulation of the levels of the cytokines was demonstrated in the group treated with 100 mg/kg G. Lucidum
. This further indicates the hepatoprotective efficacy of G. Lucidum.
The above result corroborates with the histopathological finding as shown in Figure 3
as rats administered FA show a diffuse periportal cellular infiltration with severe congestion indicating hepatic damage; however, G. lucidum
was able to reverse this effect [51