Acute safety assessment of lithium bioacummulated in mycelial biomass of Ganoderma lucidum Avaliação aguda da segurança do lítio bioacumulado em biomassa micelial de Ganoderma lucidum

Lithium is a first-line treatment for bipolar disorder, a disorder that is characterized by alternating manic and depressive episodes. However, because of its narrow therapeutic index, lithium can cause adverse effects. The association of lithium with more complex molecules, such as proteins and polysaccharides, may help control the release of lithium throughout the digestive system, thereby reducing the doses that are used by patients and consequently reducing adverse effects. The present study evaluated the acute oral toxicity of lithium bioaccumulated in mycelial biomass of Ganoderma lucidum, a research step that precedes preclinical efficacy studies. The study followed Organization for Economic Cooperation and Development guideline. Male Wistar rats (n = 6) were acutely treated by gavage with fixed doses (300 and 2000 mg/kg) of lithium bioaccumulated in mycelial biomass of G. lucidum. Changes in clinical signs, body mass, relative organ mass, biochemical parameters, blood count, leukocytes, and histopathological changes were evaluated. A group of animals that were treated with vehicle (n = 6) served as the basal group. No


INTRODUCTION
Ganoderma lucidum (Curtis) P. Karst. (Basidiomycotaete: Polyporales) has the ability to bioaccumulate metals of nutritional and pharmacological interest in its mycelial structures (MENIQUETI et al., 2020;SCHEID et al., 2020;UMEO et al., 2020;MATUTE et al., 2011;MENIQUETI et al., 2021). The bioaccumulation of metals in basidiomycetes is a particularly interesting strategy to control the solubility and availability of metals. This occurs through the association of ions with complex organic compounds, such as proteins and polysaccharides, that are slowly digested and release minerals throughout the digestion process (FARIA et al., 2019).
One of these metals, lithium, is a first-line treatment for bipolar disorder, which exerts preventive actions against suicide (CHI-TSO and CHUANG, 2011). Bipolar disorder is a chronic or episodic mental disorder (i.e., it may occur only occasionally or at irregular intervals). It is characterized by manic and depressive phases that can cause unusual, often extreme, and fluctuating changes in mood, energy, activity, and concentration or focus. It is a common, serious, chronic, and often life-threatening disease that affects 0.5-1% of the population worldwide (CARDOSO et al., 2015).
Lithium carbonate has been the first treatment choice for this psychiatric illness.
It is absorbed in the gastrointestinal tract and excreted by the kidneys (WARING et al., 2002). The treatment concentration of lithium can vary from 0.5 to 1.2 mM, but its mechanism of action has caused doubts because of its narrow therapeutic index. Another concern of lithium is its effects on kidney function, which may be impaired during treatment. The therapeutic plasma concentration of lithium is normally between 5.6 and 8.4 mg/L, and its toxic concentration is above 10.5 mg/L (SMITH and CIPRIANI, 2017).
Based on the importance of lithium for the treatment of psychiatric diseases, its bioavailability and effectiveness need to be controlled. One promising option may be its bioaccumulation in basidiomycetes. The solubility of lithium can be controlled when it is associated with organic macromolecules, such as polysaccharides and proteins, which can assist with controlled release and make treatment safer (AIDA et al., 2009). Thus, lithiumenriched fungal biomass could be a supplementation option for the treatment of bipolar mood disorders. However, despite the relevance of lithium to the treatment of psychiatric diseases and the potential production of lithium bioaccumulated fungi as a source of lithium, no studies have investigated the toxicity of lithium bioaccumulated in a myiceliall biomass of G. lucidum, which was the goal of the present study.

-Lithium bioaccumulated in a mycelial biomass of Ganoderma lucidum
Ganoderma lucidum (U17-1) from the Culture Collection of the Molecular Biology Laboratory, Universidade Paranaense (Umuarama, PR, Brazil), was used. This strain was registered (code no. A04E776) in the Sistema Nacional de Gestão do Patrimônio Genético e Conhecimentos Tradicionais Associados (SisGen), Brazil. The mycelium was added to malt extract agar (20 g/L), previously autoclaved at 121ºC for 20 min, and grown in the dark at 28ºC ± 1ºC for 10 days. The homogeneous mycelia without sectorization was selected as the inoculum. Erlenmeyer flasks that contained liquid culture medium based on sugarcane molasses (20 g/L) were autoclaved at 121°C for 20 min. Afterward, a solution of lithium carbonate (Reatec®) that was previously sterilized was added to the medium to obtain a final concentration of 30 parts per million (ppm) of lithium. Each Erlenmeyer flask was inoculated with three disks (0.5 mm diameter) of the culture medium that contained the mycelia. Mycelial biomass growth occurred at 28ºC ± 1°C for 21 days in the dark, without shaking. The mycelial biomass was dehydrated at 60°C until a constant mass was obtained. Lithium extraction followed the methodology of Yokota et al. (2016). The determination of lithium concentrations in the samples was performed by atomic flame absorption spectrophotometry (GBC model 932 plus). For lithium quantification, a calibration curve was used (R2 = 0.998) with an analytical standard (IAA-203 Lot K00461 ultra scientific analytical solutions) with a detection limit of 0.01 mg/ml. The concentration of lithium per gram of biomass was 246.81 µg.

-Experimental animals
The acute toxicity of lithium accumulated in mycelial biomass was evaluated in male Wistar rats of 300-400 g, obtained from the State University of Maringá (UEM).
The animals were housed in the vivarium of the UNIPAR Neuroscience Laboratory with free access to a liquid and solid diet under controlled environmental conditions (temperature: 20ºC ± 2°C; relative humidity: 50% ± 10%; 12 h/12 h light/dark cycle). The

-Experimental design
The animals were randomly assigned to groups (n = 6/group). The rats' group assignments were marked on the tail. The rats were housed in rectangular polypropylene boxes for 10 days before beginning the experiments to allow acclimatization to the laboratory conditions. The lethal dose (LD50) was determined according to Organization for Economic Cooperation and Development (OECD) guideline 423. Groups of animals were dosed in a stepwise manner using fixed doses of 5, 50, 300, and 2000 mg/kg. The initial dose was 300 mg/kg because there is no information on a substance to be tested.
Other groups of animals were dosed with higher or lower fixed doses, depending on the presence or absence of signs of toxicity or mortality. This procedure was performed until the dose that caused evident toxicity or not more than one death was identified, when no effect was observed at the highest dose, or when deaths occurred at the lowest dose. After an 8 h fast, the lithium bioaccumulated in mycelial biomass was administered orally by gavage in a single dose (Fig. 1). Following treatment, the rats were fasted for an additional 4 h. One group of animals (n = 6) that was treated with vehicle (deionized water) served as the basal group. substance is based on the mortality and/or moribund status of the animals, and 2-4 steps may be necessary for such an evaluation. The starting dose (300 mg/kg in this study) is tested once. If 2-3 animals died, then this was followed by testing a lower fixed dose. If one or no animals die, then the starting dose is tested twice, and a higher fixed dose level is used in the next step until the appropriate Globally Harmonized System category of toxicity is reached.

-Assessment of clinical signs
After administering the lithium bioaccumulated in mycelial biomass of G. lucidum, the animals were monitored during the first 30 min and then 1, 2, 3, and 4 h after treatment. Behavioral parameters and clinical signs (i.e., self-cleaning, piloerection, dyspnea, abdominal constriction, diarrhea, prostration, ataxia, sedation, coma, and death) were recorded according to the method of Almeida et al. (1999). After the first 4 h of observation, the animals received water and food and were observed daily for 14 days to record clinical changes and deaths.

-Euthanasia and material collection
On day 15, after fasting for 12 h, the rats were euthanized by deep anesthesia with isoflurane in a saturation chamber (1-3%). Blood was collected by decapitation. Levels of cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, urea, and creatinine were determined using commercial kits in an automated system. Red blood cell counts, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, leukocytes, rods, lymphocytes, and platelets were recorded. The liver, spleen, heart, and kidneys were removed, carefully dissected, and weighed on an analytical balance. For kidney mass, the average mass between the right and left sides was used. The mass of each organ was multiplied by 100 and divided by the animal's body weight before euthanasia to obtain the relative organ mass (%).

-Histopathological analysis
Fragments of the liver, spleen, heart, kidney, and brain were carefully removed, washed with ice-cold saline, and packed in 10% buffered formalin. After standard histological processing, the sections were stained with hematoxylin and eosin and histopathologically analyzed by a pathologist veterinarian. The slides were analyzed under a Leica DM 2500 optical microscope to detect cellular changes that resulted from the treatments according to Lima et al. (2019) and Souza et al. (2020).

-Statistical analysis
The data were first analyzed for homogeneity of variance and a normal distribution. One-way analysis of variance (ANOVA) was then used, followed by the Newman-Keuls post hoc test. The level of significance was 95% (p ≤ 0.05). For the statistical analysis and generation of the graphs, GraphPad Prism 6.0 software was used.

DISCUSSION
The present study tested the acute toxicity of lithium bioaccumulated in mycelial biomass of the Ganoderma lucidum in Wistar rats as proposed by OECD guideline 423.
No changes in body mass or relative organ mass were observed in the animals, with no changes in biochemical, hematological, or histopathological parameters. Our findings indicate that acute oral treatment with 300 and 2000 mg/kg of this substance is nontoxic over 14 days in rats when administered at single dose.
In preclinical toxicology studies, hematopoietic evaluations are recommended as an important way to evaluate the toxicity profile of a substance and as a pathological index (ADENEYEA et al., 2006). Several biochemical parameters are important for explaining the toxicity of test substances (PETTERINO and ARGENTINO-STORINO, 2006). The profile of liver function (aspartate and alanine aminotransferase) and kidney function (urea and creatinine) are important predictors of the possible hepatic and renal toxicity of drugs (HILALY et al., 2004). Hematopoietic and biochemical changes can be reflected by cellular changes, assessed by histopathological examination (MATSUZAWA et al., 1995). In the present study, no alterations of hematological, biochemical, or histopathological parameters were found in rats that were acutely treated with lithium bioaccumulated in mycelial biomass of G. lucidum bioaccumulated with lithium at the 2000 mg/kg dose, indicating that this substance is nontoxic to the hematopoietic system, including the liver, kidneys, spleen, and heart. These results suggest possibilities of further preclinical studies of the effectiveness of this natural product with bioaccumulated lithium in animal models of anxiety, depression, and bipolar disorders.
The use of lithium for the acute treatment of bipolar disorder involves several aspects. The mood phase of bipolar disorder can move to the depressive phase before the euthymic state is reached. One disadvantage of lithium is that it can cause adverse effects, such as drowsiness, psychomotor retardation, and akathisia, which can mimic changes in mood (YILDIZ et al., 2011;KIM et al., 2017). Lithium carbonate has been the drug of choice for the treatment of bipolar disorder and is absorbed in the digestive system. For therapeutic purposes, it is necessary to reach a plasma concentration between 5.6 and 8.4 mg/L. The toxic concentration of the drug is above 10.5 mg/L, thus indicating its narrow therapeutic index (ORUCH et al., 2014;SMITH and CIPRIANI, 2017). However, the adverse effects of lithium are dose-dependent. A lower dose of lithium restricts its adverse side effects (ALBERT et al., 2014).
One of the difficulties in using lithium is the therapeutic index for treatment. The association of lithium with more complex molecules that are present in basidiomycetes is one interesting strategy to control the solubility and availability of lithium, which may help control release of the ion throughout the digestive system and thus mitigate the Science continually works to develop alternative strategies to decrease the number of animals that are used in toxicity studies and improve predictability and safety assessments (PRIOR et al., 2020). However, although new approaches and alternatives are growing for screening purposes, many components of regulatory toxicological assessments still depend on animal research, which is likely to continue into the foreseeable future until more reliable alternatives are discovered and validated as substitutes for animal use, especially in pharmacokinetic studies of absorption, distribution, metabolism, and excretion (GOH et al., 2015). Although animal toxicity studies remain necessary, the 3Rs (Reduce, Refine, Replace) of animal research are still applicable. Any data that are generated from animals must be relevant and used to minimize the risk of adverse effects in humans. Currently, application of the 3Rs in toxicological studies includes refinements in research projects that seek to promote animal welfare and consequently obtain more reliable data (SPOONER et al., 2019, KENDRICK et al., 2020, in addition to reducing the number of animals that are used by optimizing the size of the experimental groups (CHAPMAN et al., 2013). Thus, the correct conduct of toxicity studies can positively influence both preclinical and clinical investigations of the efficacy of new molecules, leading to a greater likelihood of translational success and reducing the final friction in the final stages of drug development (PRIOR et al., 2020).

CONSLUSION
Lithium bioaccumulated in mycelial biomass of the Ganoderma lucidum did not induce signs of toxicity in rats that were acutely orally treated. However, further studies that test multiple dosing of lithium bioaccumulated in mycelial biomass of G. lucidum are required to better determine the comprehensive toxicity profile of this substance.