Toxic metabolite profiling of Inocybe virosa

Wild mushroom foraging involves a high risk of unintentional consumption of poisonous mushrooms which is a serious health concern. This problem arises due to the close morphological resemblances of toxic mushrooms with edible ones. The genus Inocybe comprises both edible and poisonous species and it is therefore important to differentiate them. Knowledge about their chemical nature will unambiguously determine their edibility and aid in an effective treatment in case of poisonings. In the present study, the presence of volatile toxic metabolites was verified in Inocybe virosa by gas chromatography. Methyl palmitate, phenol, 3,5-bis (1,1-dimethyl ethyl) and phytol were the identified compounds with suspected toxicity. The presence of the toxin muscarine was confirmed by liquid chromatography. The in vitro study showed that there was negligible effect of the digestion process on muscarine content or its toxicity. Therefore, the role of muscarine in the toxicity of Inocybe virosa was studied using a bioassay wherein metameters such as hypersalivation, immobility, excessive defecation, heart rate and micturition were measured. Administration of muscarine resulted in an earlier onset of symptoms and the extract showed a slightly stronger muscarinic effect in comparison to an equivalent dose of muscarine estimated in it. Further, the biological fate of muscarine was studied by pharmacokinetics and gamma scintigraphy in New Zealand white rabbits. Significant amount of the toxin was rapidly and effectively concentrated in the thorax and head region. This study closely explains the early muscarinic response such as miosis and salivation in mice. By the end of 24 h, a relatively major proportion of muscarine administered was accumulated in the liver which stands as an explanation to the hepatotoxicity of Inocybe virosa. This is one of the rare studies that has attempted to understand the toxic potential of muscarine which has previously been explored extensively for its pharmaceutical applications.

GC-MS analysis. GC-MS analysis of the hydro-ethanolic extract of Inocybe virosa was performed using the method followed by Sai Latha et al. 15 . Agilent GC 6890 N JEOL GC Mate II with Ion Trap gas-chromatography equipped with HP5 capillary column (30 m × 0.32 mm; coating thickness 0.25 μm), interfaced with Agilent 240 MS Ion Trap mass detector was used for this analysis. The analytical conditions were as follows: injector temperature-20 °C; transfer line temperature-250 °C; oven temperature programmed from 50 °C to 250 °C at 10 °C /min; helium as the carrier gas at 1 mL/min; ionization voltage-70 eV; ion source temperature-250 °C; interface temperature-250 °C; mass range-50-600 mass units. The identification of components was performed by comparison of their linear retention indices and by matching against commercial mass spectra libraries from NIST and MS literature data.

RP-HPLC and LC-MS analysis.
The Inocybe virosa mushroom as well as its hydro-ethanolic extract were analysed for the presence of muscarine using Reverse Phase-High Pressure Liquid Chromatography (RP-HPLC). The samples were subjected to extraction procedure followed by Yoshioka et al. 16 with slight modifications. 0.2 g of finely powdered samples was homogenized with 2.5 mL 0.5% formic acid in methanol and sonicated for about 5 min. This mixture was centrifuged at 1,000 ×g for 5 min and the supernatant was collected. The final volume was adjusted to 5 mL using 50% methanol (V/V). For the clean-up, 1 mL was loaded to Oasis HLB cartridge preconditioned with methanol. The first 0.5 mL of the elute was discarded and the remaining was collected for analysis. Muscarine was determined using JASCO HPLC system (Japan) consisting of quaternary pump and PDA (Photo Diode Array) detector. Separation was carried out on a HiQSiL Reverse phase C 18 column (250 mm × 4.6 mm, particle size 5 µm). The absorbance was monitored at 235 nm and the results were analysed using LCNET software. A gradient elution system using the mobile phase A (5 mM K 2 HPO 4 buffer, pH-2.5 prepared in Milli-Q water) and mobile phase B (Methanol, HPLC grade) at a flow rate of 0.6 ml/min. The gradient program was as follows: (1) 0 min-100% A, (2) 3 min-40% A, (3) 9 min-40% A, (4) 12 min-100% A. The muscarine content was estimated in the mushroom extract by using standard muscarine (Sigma-Aldrich, Chemical purity ≥ 98%). The chromatogram of RP HPLC of standard is shown in Supplementary Fig. S1.
An Agilent HPLC 1260 Series connected with Shimadzu LC-MS 2020 system and an Agilent Eclipse plus column (4.6 × 250 mm, 5 µm) was used for MS analysis of the samples. The same gradient program was employed for the analysis as in HPLC with a slight modification of replacing K 2 HPO 4 buffer with 0.1% formic acid as solvent A. The specifications of MS analysis were as follows: ESI-Positive ionization; Desolvation line temprature−250 °C; Heat block temperature−200 °C; Scan-50 m/z to 500 m/z; Scan speed-455u/s; Detector Voltage-0.8 kV; Mobilizing gas flow-1.5 L/min.

Cytotoxicity evaluation of Inocybe virosa. Cell lines. The immortalized cell lines, NCM460 and
Chang liver cell line were both obtained from National Centre for Cell Sciences (NCCS), Pune, India and grown at 37 °C in a humidified atmosphere of 5% CO 2 using MEM (Minimum Essential Media), supplemented with 10% FBS and antibiotic solution. About 80% confluent cells were used for the assays. The analysis was carried out in triplicates.
Cell viability. The in vitro cytotoxicity of the mushroom extract was assessed on NCM460 and Chang liver cell lines using the dye 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The cells (100 μL; 1 × 10 5 cells/mL) were seeded into 96 well plates and incubated for 24 h. The medium was then removed from the wells and replaced with 100 μL filter sterilized medium containing the mushroom extract. The cells were incubated with different concentrations (0-2.5 mg/mL) of the extract for the next 24 h. MTT (100 μl; 0.5 mg/ mL) was added to each well and further incubated for 2 h. Subsequently, the medium was removed and DMSO (100 μL) was added. The absorbance was recorded at 570 nm 17 and cell viability was expressed with respect to control. The toxic effects of the extract on cell morphology were also noted and photographed.
Evaluation of in vivo oral toxicity of Inocybe virosa. Animals and housing conditions. Female Balb/c mice of body weight 25-30 g, from the Central Animal Facility of the laboratory (Defence Food Research Laboratory), were used for the study. Six animals per cage were housed in standard sized cages with corn cob bedding and maintained under controlled 12 h light/dark cycle, optimum conditions of temperature and relative humidity (23 ± 2 °C, 40-60% humidity). Potable water and pellet diet were provided ad libitum to the animals. The experimental procedures were performed in accordance with the guidelines of CPCSEA ( Dose formulation and administration. The mice were randomly divided into 3 groups (n = 6). The animals were fasted for 2 h prior to the administration of mushroom extract, and control animals were administered water in place of the extracts. For acute toxicity study, a single dose of each mushroom extract was administered to two groups of animals, one at 250 mg/kg body weight (bwt) and the other at 500 mg/kg bwt. The animals were observed for any abnormal behavioural responses and body weight changes. Three animals of each group were sacrificed after 24 h and the remaining three animals were sacrificed on day 14 under anesthetic conditions. Blood was drawn through cardiac puncture and analysed for haematological parameters and biochemical parameters were analysed in the serum. The organs were collected for histopathological analysis.
Blood biochemical analysis. Biochemical parameters in serum were analyzed to assess the liver and kidney functioning using the commercially available kits from Agappe. The liver function tests (LFTs) included the estimation of enzyme activities such as aspartate transaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALK), and the total bilirubin (TBIL) content. The kidney function tests (KFTs) included the estimation of creatinine (CRE), blood urea nitrogen (BUN), and total protein (TPR).
Histopathological studies. The organs viz., brain, kidney, liver, small and large intestine were excised on sacrificing the animals. The tissues were gently rinsed in phosphate buffer (pH 6.8) and were fixed with 10% neutral buffered formalin solution. The tissues were then embedded in paraffin wax, sectioned using a microtome and mounted onto glass slides. The paraffin was cleared and this was followed by staining the tissue section with hematoxylin and eosin. The cross sections of the tissues were observed under a compound light microscope at 20 × objective. Toxicity of muscarine. In vitro digestion of muscarine. In vitro digestion of Inocybe virosa extract was carried out using the model proposed by Soler-Rivas et al. 18 . 10 g of the extract was mixed with 5 mL of water and heated to boiling. The sample was cooled and mixed with 1 mL of healthy volunteer's saliva and 1 mL of phosphate buffer (0.08 M, pH 6.7). The pH was then set to 2 with 6 M HCl and pepsin was added (5 mg per gram homogenate). The mixture was then incubated for 2 h at 37 °C with slow stirring at 60 rpm in a rotary shaker. The sample was subjected to further digestion by adding 2 mL of pancreatic solution (4 mg pancreatin and 25 mg bile salts per mL of 0.1 M NaHCO 3 ) and the pH was adjusted to 7.5 with NaOH. To the digested samples, 1 g of NaHCO 3 was added for maintaining the pH. The mixture was incubated again for 2 h at 37 °C with slow stirring at 60 rpm.
On complete digestion of the sample, cytotoxicity of the digestate was assessed on a human epithelial colorectal cell line, Caco2 obtained from NCCS, Pune. The cells were grown and maintained using Minimum Essential Medium (MEM) (Sigma-Aldrich) supplemented with 10% foetal bovine serum (HiMedia), 1% penicillin-streptomycin (Sigma-Aldrich), 1.5 g/L Sodium bicarbonate and 110 mg/L Sodium pyruvate (Sigma-Aldrich) at 37 ºC in a humidified atmosphere containing 5% CO2. The cells (100 μL; 1 × 10 5 cells/mL) were seeded into 6 well plates and incubated for 24 h. On the adherence of cells, medium was removed from the wells and the wells in triplicates were incubated overnight with 500 μL filter sterilized medium containing the mushroom extract and digestate. The cytotoxic effect was evaluated using the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). MTT (100 μl; 0.5 mg/mL) was added to each well and further incubated for 2 h. Subsequently, the medium was removed and DMSO (100 μL) was added. The absorbance was recorded at 570 nm 17 and cell viability was expressed with respect to control. The toxic effects of the extracts on cell morphology were also noted and photographed. A set of cells in triplicates were also exposed to the mixture of digestive enzymes used for the in vitro digestion. To verify the effect of digestion on muscarine, the digestate was analysed for the toxin concentration using RP-HPLC method detailed in "RP-HPLC and LC-MS analysis" section. . Another set of mice were administered an equivalent dose of standard muscarine (MC) (i.e., 4.5 mg/kg bwt muscarine corresponds to the amount present in 500 mg/kg bwt of IV (Inocybe virosa) extract based on the HPLC analysis) and water was administered to the control group. The behavioural pattern of the animals was observed and three notable metameters i.e., hypersalivation, immobility and excessive defecation were scored in each animal by placing it individually in a box with grids (5 cm × 5 cm) 19 . Salivation was scored using the following numerical scale: 0 = Absence of response, 1 = Jaw and fur noticeably wet but no drooling noted, 2 = Maximal response with saliva actively dripping from the jaws. Immobility of the animal was scored using the following scale: 0 = Free movement of the animal, 1 = Movement of the animal restricted to 3-6 squares, 2 = Movement of the animal restricted to less than 3 squares and defecation was scored as 0 = normal, 1 = mild, 2 = severe. The sum of the scores for each metameter at regular intervals as well as a cumulative score was used to compare the effect of muscarine with that of the extract. Another set of animals (n = 3) were studied for the effect of muscarine and extract on heart and bladder in terms of heart rate and extent of micturition respectively. Each mice was placed individually on dried filter papers immersed in alkaline phenol-red and total area of urine spots was measured to indicate the extent of micturition 20 . Heart rate was measured using the blood pressure monitoring machine (Muromachi Kikai Co. Ltd).
Pharmacokinetics and biodistribution studies of radiolabelled muscarine. Radio  ) at the hydroxyl groups using stannous chloride as the reducing agent 21 . Briefly, 100 μL of 99m TcO 4 − (approximately 5 mCi, obtained by solvent extraction from Molybdenum) was mixed with stannous chloride solution (200 μg) in 10% acetic acid to reduce the Technetium. The pH was adjusted to 6.5-7.0 using 0.5 M sodium bicarbonate. To this, 0.5 mg of MC was added and incubated for 15 min at room temperature. The labelled formulation thus obtained was stored in sterile evacuated sealed vials for subsequent studies.
Radio labelling efficiency. Radiolabelling efficiency (LE) of technetium labelled muscarine chloride formulation was determined by ascending instant thin layer chromatography (ITLC) using silica gel (SG)-coated fibre glass sheets, approximately 10 cm in length (Gelman Science Inc., Ann Arber, MI, USA). ITLC was performed using acetone as the mobile phase. 2-3 μL of radiolabelled formulation was applied at a point of 1 cm from one end of an ITLC-SG strip. The strip was developed in acetone and solvent front was allowed to reach approximately 8 cm from the origin. The strip was cut into two equal halves and radioactivity in each segment was determined in a gamma-ray counter (Caprac Capintech Inc., NJ. USA). The free 99m TcO 4 moved with the solvent (R f = 0.9) while radiolabelled formulation remained at the point of application. Percent labelling efficiency was calculated as follows: where T is radioactive counts at top and B is the radioactive counts at bottom.
In vitro stability of radio labelled formulations. The in vitro stability of radio labelled muscarine chloride formulation was determined by mixing 100 μL of radio labelled formulation with 1.9 mL of human serum and incubating it at 37 °C. Any change in radio labelling efficiency was monitored at regular intervals over a period of 24 h by ITLC as described previously.
Pharmacokinetics and biodistribution of muscarine. The study was performed in normal, healthy, female New Zealand rabbits (n = 3) weighing 2.5-3 kg [22][23][24] . Animals were injected with 1.5 mCi 99m TcO 4 − labelled muscarine formulation through the dorsal ear vein. Blood (0.2 mL) was withdrawn through the vein of another ear at periodic intervals in pre-weighed tubes. The weight of the blood withdrawn at each interval was noted and the radioactivity measured using a gamma-ray spectrometer (Caprac Capintech Inc., NJ, USA). The activity present in total blood was calculated by considering 7.3% of total body weight as total blood volume. Static scintigraphy images were acquired at periodic intervals on administration of the radiolabelled formulation by positioning the animal under the gamma camera. The results were expressed as percentage of the injected dose.

Results and discussion
Volatile compounds of Inocybe virosa. The presence of volatile toxic metabolites in Inocybe virosa was verified using gas chromatography. Figure 1 presents the gas chromatogram of hydro-ethanolic extract of Inocybe virosa. The compounds were identified on the basis of their retention times. Peak assignment was confirmed using NIST library of GC-MS. The molecular structures and molecular formulae of the compounds as shown in Fig. 2 were obtained based on the MS fragmentation pattern. The volatile profile of the extract showed a majority of fatty acid esters: E-6-tetradecen-1-ol acetate, hexadecanoic acid-methyl ester, heptadecanoic acid, 16-methylmethyl ester, 9,15-octadecadienoic acid-methyl ester (ZZ), nonadecanoic acid-18-oxo-methyl ester. A previous study has also shown that fatty acid esters constituted a major fraction of mushrooms such as Pleurotus eous 25 . Among the esters identified, hexadecanoic acid, methyl ester (methyl palmitate) was reported to be associated www.nature.com/scientificreports/ with acaricidal or insecticidal activity 26 and has shown to cause neurotoxicity in mites 27 . Other constituents identified in the extract were phenol, 3,5-bis(1,1-dimethyl ethyl), Phytol and Flavone. It has been reported that flavone is associated with nematicidal effects 28 . Phenolic toxicity has been demonstrated on human colonic epithelial cells 29 and is identified as an environmental pollutant affecting aquatic macrophytes 30 . It can be speculated that these compounds may have possibly contributed to the toxicity of Inocybe virosa, although a more detailed study is required to ascertain this inference. It is reported that the volatile composition is independent of the  www.nature.com/scientificreports/ mushroom state (fresh, frozen or dried) 31 and therefore, the volatile secondary metabolites of wild mushroom species can serve as biomarkers for species discrimination as well 32,33 .
Identification of muscarine in Inocybe virosa. The symptoms of muscarinic syndrome observed in mice on administration of Inocybe virosa extract (Fig. 3) was an indication to verify the presence of muscarine in this mushroom. Inocybe species are known to contain muscarine and its detection methods have varied widely over the years. Chemotaxonomic tests 34 , paper chromatography using Thies and Reuther's reagent 35 and gas chromatography 11 were used to identify muscarine in Inocybe species. Liquid Chromatography methods were developed and coupled to mass spectrometry for specific and rapid identification of muscarine in matrices such as food 36 and urine 37 . The procedure given by Yoshioka et al. 16 was found best suited for our laboratory settings. However, 0.1% (v/v) formic acid (Mobile phase A) was replaced by K 2 HPO 4 buffer to obtain a better resolution and sharper peaks. The procedure was standardized by varying the concentration and pH of the buffer. The salt concentration was maintained at minimal levels to prevent any interference (5 mM and 10 mM), while not compromising on the resolution of chromatographic peaks. pH variations ranged from acidic to neutral (2.5, 5 and 7) as the compound of interest was cationic in nature. The combination of 5 mM at pH 2.5 was found optimum for detection of muscarine. This is one of the first reports of the occurrence of muscarine in Inocybe virosa. The estimated toxin content was 0.27 mg/g in the extract (Fig. 4). It was evident that hydro-ethanolic extraction increased toxin concentration approximately ten folds in comparison to the crude methanolic extraction. Our HPLC finding was further validated by LC-MS analysis based on its molecular weight (174 g/mol) (Fig. 5). Our results reiterate the point made by Kosentka et al. 38 that the occurrence of muscarine in species of the genus Inocybe is an ancestral trait. From these results, it can be clearly inferred that the manifestation of muscarinic symptoms was an outcome of the presence of muscarine in the mushroom.
In vitro toxicity of Inocybe virosa. Effect on cell viability. On 24 h exposure to the mushroom extract, there were distinct cytotoxic effects on the morphological structure of the NCM460 intestinal cells (Fig. 6). The cell viability showed a significant (p < 0.05) reduction in case of the mushroom extract with respect to control. Similarly, in case of the Chang liver cells, a dose-dependent decrease in cell viability was observed on exposure to different concentrations of the extract (Fig. 7) as it was noted in the intestinal cells. Inocybe virosa was toxic to hepatocytes with a low IC 50 value (0.18 mg/mL). For the preliminary in vitro study, NCM460 colon epithelial cell line was selected to represent the intestinal barrier as these cells are the earliest to come in contact with the mushrooms on their consumption. The toxic potential of the mushroom was assessed considering their effects on cell morphology and cell viability. Morphological changes and abnormalities such as cell shrinkage and distortion in cell shape were evident in the cells exposed to the extract as shown in Fig. 6. Inocybe virosa had toxic effect on intestinal cells showing a significantly low IC 50 value of 0.63 mg/mL. This indicated a marked possibility of intestinal toxicity associated with their consumption. Our finding of intestinal toxicity associated with Inocybe virosa stands in agreement with the severe gastrointestinal symptoms documented on consuming this mushroom [39][40][41] .
Liver being an organ of defense against the exogenous compounds, an in vitro toxicity evaluation using the representative Chang liver cell line was considered in the study. Cell viability was monitored to assess the overall effect on the cells. As evident from Fig. 7, the mushroom extract exhibited potential hepatotoxicity. A previous study by Kawaji et al. 42 demonstrated in vitro hepatotoxicity on exposure to toxic Amanita species such as Amanita abrupta, Amanita virosa, and Amanita volvata. This is also one of the first reports of in vitro toxicity in liver cell lines w.r.t Inocybe virosa.   www.nature.com/scientificreports/ In vivo toxic effects of Inocybe virosa. General observations. On oral administration of the mushroom extract at the two selected doses in acute toxicity evaluation, the animals showed no mortality during the study period. Also, the administration of extract caused no significant reduction in body weight of the animals in comparison to control. However, muscarinic symptoms viz., excessive salivation, lacrimation, and urination were markedly observed in a dose-dependent manner.    (Tables 2 and 4). The levels of the liver enzymes though slightly lowered by day 14, they were significantly higher in case of Inocybe virosa at 500 mg/kg bwt (Table 3). There were degenerative changes in kidney at both doses after 24 h of administration of Inocybe virosa. At 500 mg/kg bwt, glomerular and tubular distortion with foci of haemorrhage was seen while the damage was milder at 250 mg/kg bwt. The tissue distortion at higher dose of Inocybe virosa was observed on day 14 also (Figs. 8 and 9). With respect to liver histology, Inocybe virosa, at both the doses caused hepatic lobular distortion after 24 h and a mild feathery degeneration was observed on day 14 at higher dose only (Figs. 10 and 11). Inocybe virosa extract showed mild hyperplasia at the lower dose while at 500 mg/kg bwt, sloughing of mucosa with non-specific inflammation and distortion of glands w.r.t intestinal tissue after 24 h. There was no intestinal pathology noted on day 14 (Figs. 12 and 13). The brain histology however showed no obvious pathology.
In vivo acute toxicity study was useful in verifying if an oral consumption of the selected mushroom Inocybe virosa caused any in vivo systemic toxic effects and also to identify if there was any specific target organ. Female mice were preferred for the study as they are generally more sensitive to toxic effects 43 . As the most significant human exposure to mushrooms occurs through ingestion, oral route was chosen for this study. The yield on extraction was about 3.5% of fresh weight of the mushrooms and therefore a dose of 500 mg mushroom extract/ kg body weight (bwt) for mice (25-30 g) when extrapolated to humans (60 kg) is approximately equivalent to one serving of mushroom per meal with conversion factor of 1/12 44 . The OECD guideline TG 423 (2001) for acute toxicity was followed but at doses of the mushroom extract which had relevance to daily intake of mushrooms by humans. The onset of symptoms on mushroom poisoning is usually within 6-24 h 45 . However, the clinical manifestations in many poisonings may also have a latency period up to 14 days 46 . Therefore in our study, 24 h and 14 days were the time points considered to evaluate toxic effects in response to a single oral administration of the mushroom. Considering the hematological parameters, increased level of WBCs (Table 1)   www.nature.com/scientificreports/    www.nature.com/scientificreports/ inflammatory response. A report of leucocytosis (an increase in WBC count) on account of wild mushroom poisoning in Iran 47 supports our finding. In addition to hematological parameters, clinical biochemistry data also plays an important role in determining toxic effects. As mushroom poisonings are generally associated with gastrointestinal, renal, hepatic, or neurological effects 48 , small and large intestines, kidney, liver and brain were examined for pathological signs. ALT and AST present in cytoplasm of hepatocytes are sensitive markers of liver tissue damage, as they show a marked elevation in their activities in the bloodstream on account of any liver injury 49 . ALP which is also particularly concentrated in liver shows an increase in case of distortion in hepatic architecture. The biochemical parameters ALP, ALT, AST including total bilirubin are therefore reliable indicators of hepatic functions; while total protein, creatinine, and urea levels are good measures of renal functions 50 . The mushroom Inocybe virosa, increased levels of ALT, AST, ALP and TBIL after 24 h (Table 3) indicating altered functional status of liver with hyperbilirubinemia which correlated well with histopathological observations made in liver tissue ( Fig. 10 and  11). It is thus reasonable to speculate that increased activity of these enzymes could be due to hepatic damage and subsequent leakage of these enzymes into circulation. These sensitive biomarkers were elevated in accidental poisonings in humans with wild mushrooms 51,52 and also in case of amanitin intoxication 53 . This clinical data stands in agreement with our findings. The persistence of this damage up to day 14 in case of Inocybe virosa is of toxicological significance and strongly affirms its hepatotoxicity. There are reports of acute hepatitis on consumption of wild mushrooms 54 which substantiates the hepatotoxicity noted in our study. Pathology was noted in kidney tissue on exposure to Inocybe virosa at both doses after 24 h and at the higher dose on day 14 (Figs. 8  and 9). This was in consonance with the significant increase shown in KFTs (Table 2) indicating that kidney was also a target organ of this mushroom.
The evident toxic effects of Inocybe virosa well justify its nomenclature virosa (meaning full of poison) 55 .The typical muscarinic symptoms on the administration of Inocybe virosa speculates the presence of toxin muscarine in this new and unexplored Inocybe species. Literature indicates that the consumption of Inocybe species is associated with muscarinic syndrome 56 . Muscarine is a characteristic component of genus Inocybe, and in fact, our study affirms the presence of muscarine in Inocybaceae family as an ancestral trait 38 . Effect of in vitro digestion on muscarine. The present study also attempted to substantiate the toxic effects associated with the oral intake of the mushroom (usually as a part of diet) using an in vitro model. Therefore, the effect of digestive enzymes and the associated pH variations on muscarine content and toxicity of the extract was analysed by simulating the digestion conditions in mouth, stomach and small intestine. The effect of the digestate was studied on Caco-2 intestinal cells which were representative of the intestinal barrier 57 . The digestate represented the bioaccessible fraction which is most likely to get absorbed by the intestinal enterocytes. Its effects were assessed based on the morphological changes observed in cells exposed to it (Fig. 14) and estimated in terms of cell viability (Fig. 15). There was no significant decrease in cell viability with respect to control on exposure to the digestate control (the mixture of digestive enzymes used for in vitro digestion). Hence, it can  www.nature.com/scientificreports/ be concluded that the digestive enzymes had negligible adverse effects on the cells. In comparison to control, a significant decrease in cell viability was noted in case of digestate and extract. However, the extent of damage on exposure to extract was not significantly different from that of digestate indicating that digestion had no altering effect on the toxicity of extract. Interestingly, there was only a slight difference in the toxin content prior to (2.92 mg/g) and after (2.62 mg/g) the digestion process as shown in Fig. 16. This confirms the findings of Clark and Smith 58 that the toxin muscarine is resistant to cooking or any other means of processing. The stability of muscarine to boiling, varying pH or the action of pepsin as pointed out by Fraser, 1957 59 was also validated by this study.  www.nature.com/scientificreports/ Analysis of muscarinic response in mice. There are evidences which indicate that the consumption of Inocybe species is often linked to manifestations of the muscarinic syndrome 58,60,61 . Salivation, lacrimation, urination, diaphoresis, gastrointestinal effects and emesis are some of the symptoms of muscarinic poisoning, accompanied by miosis, bronchoconstriction and bradycardia at times 62 . Malone and Brady 63 had attempted to determine the relative muscarinic potency of Inocybe species based on these symptoms. The Inocybe mushrooms were divided into four groups based on the severity of their toxic effects 64 (i) Species with pronounced muscarinic effect (Ex: I. fastigiata), (ii) Species with weak muscarinic effect (Ex: I. umnbrina), (iii) Species that produce an unstable effect (Ex: I. jurans) and (iv) Species without any muscarinic effect (Ex: I. bongardii). Therefore, an in vivo study is required to assess the muscarinic potency of Inocybe virosa. The extract was administered at a dose which had relevance to daily intake of mushrooms by humans. A dose of 500 mg mushroom extract per kg bwt for mice when extrapolated to humans (60 kg) is approximately equivalent to one serving of mushroom per meal (Conversion factor of 1/12) 44 .It was also our interest to compare the muscarinic response in mice on exposure to the extract and the equivalent muscarine content (as estimated by HPLC) in order to assess the role of muscarine in the mushroom toxicity. The observable symptoms were hypersalivation and miosis as shown in Figs. 3A,B respectively, which appeared within 5-10 min of administering both the extract and muscarine. This clearly pointed out that muscarine is a fast acting toxin as was suggested by de Oliveira 48 . The animals also showed excessive defecation and urination with marked phases of immobility, occasionally accompanied by myoclonic seizures. The bioassay conducted by Malone et al. 19 in rat involved the measurement of tear secretion and an estimation of drooling. In this study, salivation, immobility and defecation were the selected metameters which were scored to assess the muscarinic response and the animals were observed till they returned to normalcy. The results showed that the extract had a slightly stronger muscarinic effect in comparison to an equivalent dose of muscarine considering total duration of the symptoms and also the time over which symptoms were severe (Fig. 16). However, the onset of symptoms was relatively earlier for muscarine than the extract, though not significantly different (Fig. 17). It was noted that salivation continued for significantly longer duration and the duration of severity for defecation was significantly higher for the extract in comparison to muscarine.
Muscarine is a non-selective acetylcholine receptor agonist. However, its cationic nature prevents its diffusion through the lipid barrier between the bloodstream and brain. Therefore, muscarine given in diet or injected into bloodstream has negligible effect on brain and does not affect the central nervous system 65 . The structural elements of muscarine closely resemble the active conformation of acetylcholine. Thus an efficient binding of muscarine to the acetylcholine receptors is favoured which causes profound activation of peripheral parasympathetic nervous system. Muscarine arriving at the synapse by diffusion from the blood binds reversibly to these www.nature.com/scientificreports/ receptors and turns them on and this receptor bound muscarine is not removed by acetylcholine esterase. This activation continues until the muscarine concentration falls by diffusion to a level much below the threshold and propagation of the signal is eventually terminated. Its effects are more pronounced on the M2 and M3 muscarinic acetylcholine receptors distributed on the smooth muscles and exocrine glands. Hence, muscarinic action potentiates secretions of the salivary, lacrimal and sweat glands causing characteristic symptoms viz., salivation, lacrimation and perspiration. The present study validates the findings of Lurie et al. 66 that the poisonings by Inocybe species is associated with constriction of pupils, muscle spasms, diarrhoea, slowing of heartbeat and a drop in blood pressure. In a study carried out by Ochillo et al. 67 , it was demonstrated that muscarine elicited a contraction of ileal longitudinal muscles. This could possibly explain the increase in defecation (Fig. 17) as a consequence of increased persistalsis. The muscarinic effect is also exerted on smooth muscles of heart and urinary bladder. The heart rate and extent of micturition were therefore measured which were indirect indices to assess this muscarinic effect (Fig. 18). According to the toxicological studies demonstrated by Clark and Smith 58 , Clitocybe illudens and Inocybe infida caused the characteristic muscarinic effect of decreased heart rate in frogs and turtles. This finding stands in agreement to our observation of a drop in heart rate recorded in mice (Fig. 18). Muscarine syndrome can be fatal in case of ingesting large quantities of the causative species as profound activation of peripheral parasympathetic nervous system may result in convulsions and finally death 58 . The extract showed a slightly stronger muscarinic effect in comparison to the equivalent dose of muscarine. This could possibly be due to the presence of other potentiating factors like choline which contribute to the total muscarinic effect of many Inocybe mushrooms 68 . However, the earlier onset of the symptoms in case of muscarine as compared to the extract could be due to the negative matrix effect. This interference by other compounds is usually a common hindrance in case of biological samples 69 . Pharmacokinetics and biodistribution of muscarine. Muscarine was identified as one of the major toxins in Inocybe virosa and this was also evident from the results of the bioassay. Therefore, in order to deepen our understanding about its behaviour and biological fate in an in vivo system, pharmacokinetics was carried out. Gamma scintigraphy provides a clear picture of the distribution of a compound in a living system and proves advantageous in examining such processes non-invasively in real time 70 .Technetium-99m ( 99m Tc), the www.nature.com/scientificreports/ radiotracer used in the present study, is the chemical form of sodium pertechnetate obtained from molybdenum. 99m Tc has a half-life (t 1/2 ) as short as 6 h. It emits monochromatic gamma radiations of 140 keV and one of the most widely used radiotracers in Nuclear Medicine. It exhibits a unique chemistry of forming a complex with a wide range of compounds. These properties make it an ideal choice for many applications in diagnostic, pharmaceutical and biological fields 71,72 . However, Pertechnetate ion (TcO −4 ) with an oxidation state of + 7, is chemically non-reactive and cannot complex with compounds of interest upon direct addition. Therefore, it has to be reduced to a much lower oxidation state to enable efficient labelling. Stannous chloride (SnCl 2 ) is one of the commonly used reducing agent for 99m Tc. However, Sn +2 ions tend to get hydrolyzed in aqueous solution of pH 6-7 and results in formation of insoluble colloids, which also have an affinity towards 99m Tc. To overcome this hindrance in labelling, an acid is added which inhibits hydrolysis of Sn +2 ion 73 . Labelling efficiency of 99m Tcmuscarine chloride complex was assessed by ITLC and the results (Table 4) indicated that only about 8% of the radioactive complex dissociated even after 24 h, suggesting a very good radiolabelling efficiency. Further, Tc-99m labelled muscarine chloride was incubated with human serum and 0.9% saline at 37 °C for 24 h and it was found to be a stable complex which ascertains its suitability for in vivo application (Table 5). Rabbits were injected with a calculated amount of dose intravenously and this facilitated the study of the biodistribution of 99m Tc labelled muscarine chloride.
The blood pharmacokinetics showed that approximately 42% of Tc-99m labelled muscarine reached the blood pool within 5 min after administration (Fig. 19). As indicated previously by the early onset of muscarinic symptoms in mice, this finding reiterates that muscarine is a fast acting toxin. The biodistribution pattern noted immediately after administering muscarine (Fig. 20A) indicated that the major proportion of muscarine that diffused into the organs was concentrated in the thorax region (lungs, heart, liver and stomach) of the animal (Supplementary Table. S1). It was observed that muscarine was also distributed to the regions of the upper body and this observation correlated well with the appearance of characteristic muscarinic symptoms such as miosis (constriction of the pupils) and hypersalivation (activation of the salivary glands) in the animals within 10-15 min post administration (relative percentage of biodistribution as shown in the Supplementary Table. S1). The concentration of muscarine in blood gradually declined to 30% and 17% after 15 and 45 min respectively and reached to about 12.11% by the end of 1 h (Fig. 19). The biodistribution at this point (Fig. 20B) indicated the presence of muscarine in spleen, besides the organs noted earlier. The blood concentration further declined to 9.8% at the end of 2 h and the biodistribution study recorded the presence of muscarine in bladder (Fig. 20C) suggesting the beginning of renal clearance of muscarine. The fall in concentration of muscarine in blood reached  www.nature.com/scientificreports/ 5.11% by 4 h (Fig. 20D) and at the end of 24 h, only 2.72% of muscarine was noted in circulation. Gamma scintigraphy clearly indicated that the residual muscarine in the body was deposited in the liver (Fig. 20 E). Thus, direct radiolabeling using technetium-99m was found useful to study the biodistribution of muscarine. Significant amount of the toxin was quickly and effectively concentrated in the thorax and head region. This study closely explains the early muscarinic response such as miosis and salivation in mice. Also, a relatively major proportion of the muscarine administered was accumulated in the liver by the end of 24 h which possibly could have contributed to the hepatotoxicity of Inocybe virosa (Results unpublished).

Conclusion
The potential risks involved in consuming Inocybe virosa has been clearly indicated by its in vitro and in vivo toxicological evaluation. The analysis of the volatile profile of the extract indicated the presence of potentially toxic compounds such as methyl palmitate, phenol, 3,5-bis(1,1-dimethyl ethyl). However, their role in toxicity of Inocybe virosa has to be verified. This study also identified muscarine as one of the major toxin in Inocybe  Figure 19. Blood kinetics of 99m Tc-labelled MC in rabbit: 42% of Tc-99m labelled muscarine reached the blood pool. www.nature.com/scientificreports/ virosa as indicated by HPLC analysis and further confirmed by LC-MS analysis. This was further substantiated by the bioassay which showed that the extract had comparable effects of an equivalent dose of muscarine. The biodistribution study of muscarine pointed out that liver was one of the target organs of muscarine where it persisted upto 24 h.